Dissertations / Theses on the topic 'DNA damage checkpoint'
To see the other types of publications on this topic, follow the link: DNA damage checkpoint.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'DNA damage checkpoint.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Little, Elizabeth J. "DNA damage sensors in the checkpoint response." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/289950.
Full textAbstract:
The DNA damage checkpoint response detects DNA damage and responds to the damage by promoting DNA repair, transcriptional regulation, and cell cycle arrest. Prior to the beginning of this dissertation the checkpoint sensor proteins in S. cerevisiae were identified as Ddc1, Mec3, Rad9, Rad17 and Rad24. However, none of the sensors had been shown to bind DNA directly, an anticipated function of checkpoint sensors. To characterize these proteins a biochemical approach was taken to test whether any of the checkpoint sensor proteins could detect DNA. The associated DNA binding properties of Rad24 and Rad9 were identified and characterized for the first time. Both of these checkpoint sensor proteins have an affinity for ssDNA, a common intermediate DNA structure of most DNA repair processes. In addition, the DNA damage checkpoint mutant protein Rad24-1 is defective for binding to ssDNA, suggesting that Rad24 DNA binding is required for its function in the checkpoint response. The potential exonuclease activity of Rad 17 tested using purified protein and various DNA substrates. This study was based on reports that the Rad 17 homolog Rec 1 from U. maydis is a 3'→5 ' DNA exonuclease, and genetic data that indicated that Rad 17 has a role in telomere degradation. Exonuclease assays with Rad17 protein preparations and ssDNA found an associated weak exonuclease that was not significantly above background levels. Conserved residues of Rad 17 thought to be required for exonuclease activity and checkpoint activity were mutated and studied for their affect on the DNA damage checkpoint. These studies imply that in addition to the region of Rad17 that is homologous to PCNA, the long carboxy-terminal region of Rad17 is also required for its checkpoint activity. Collectively, these studies suggest that the common DNA repair intermediate structure single-stranded DNA is recognized by multiple checkpoint sensor proteins to initiate the DNA damage checkpoint response. This suggests that the initiation of the checkpoint response is the recognition of a single DNA structure instead of the many different structures of primary DNA damage by free radicals, UV, γ-radiation, alklylation, double strand breaks, and base mismatches.
2
Ho, Chui Chui. "Characterization of the regulation of p53 and checkpoint kinases in DNA integrity checkpoints /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20HO.
Full text
3
Searle, Jennifer. "The Role of PKA in the DNA Damage Checkpoint." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1123003066.
Full text
4
On, Kin Fan. "The role of MAD2L1BP in the silencing of the spindle-assembly checkpoint and the DNA damage checkpoint /." View abstract or full-text, 2009. http://library.ust.hk/cgi/db/thesis.pl?BICH%202009%20ON.
Full text
5
Carrassa, Laura. "Molecular mechanisms regulating the G2 checkpoint induced after DNA damage." Thesis, Open University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434262.
Full text
6
COLOMBO, CHIARA VITTORIA. "New insights into the regulation of DNA end processing and DNA damage checkpoint." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241167.
Full textAbstract:
L’integrità genomica è minacciata da danni al DNA che, se non adeguatamente riparati, si convertono in mutazioni, il cui accumulo causa instabilità genomica, una tipica caratteristica tumorale.
Le cellule eucariotiche reagiscono ai danni attivando la risposta ai danni al DNA. Le rotture a doppia elica del DNA (DSB) sono tra i danni più pericolosi. In Saccharomyces cerevisiae i DSB sono principalmente riparati tramite ricombinazione omologa (HR), che sfrutta sequenze omologhe come stampo per riparare il danno. La HR necessita il processamento nucleolitico (resection) delle estremità del DSB così da generare code di DNA a singolo filamento (ssDNA). La resection inizia con un taglio endonucleolitico da parte del complesso MRX insieme a Sae2, mentre l’estensione della resection è eseguita dalle nucleasi Exo1 e Dna2.
Il checkpoint da danno al DNA è una cascata di trasduzione del segnale che blocca il ciclo cellulare così che le cellule abbiano tempo sufficiente per riparare il danno. In S. cerevisiae il checkpoint è attivato dalle chinasi Tel1 e Mec1, ortologhe di ATM e ATR umane. Una volta attivate, Mec1 e Tel1 fosforilano diversi substrati, tra cui l’adattatore Rad9 e la chinasi effettrice Rad53, che amplificano il segnale.
Sia la resection che il checkpoint devono essere finemente regolati per garantire una riparazione efficiente dei DSB, evitando di generare troppo ssDNA, e per coordinare la riparazione con la progressione del ciclo. In questa tesi di dottorato, abbiamo dimostrato un nuovo livello di regolazione della resection, basato sul controllo della quantità di Exo1 da parte della proteina che lega l’RNA (RBP) Npl3. Inoltre, abbiamo studiato il ruolo di Sae2 nella riparazione dei danni e nell’attivazione del checkpoint.
Npl3 svolge un ruolo chiave nel metabolismo degli RNA ed è molto conservata nell’uomo. Poiché studi recenti mostrano forti connessioni tra metabolismo degli RNA e mantenimento dell’integrità genomica, abbiamo verificato se Npl3 fosse coinvolta nella risposta ai DSB. Abbiamo dimostrato che l’assenza di Npl3 provoca difetti nel processamento delle estremità del DSB. In particolare, Npl3 promuove la resection estesa, agendo nello stesso pathway di Exo1. Inoltre, sia l’assenza di Npl3 che l’inattivazione dei suoi domini di legame all’RNA causano una riduzione del livello di Exo1. Quindi, Npl3 promuove la resection estesa regolando EXO1 a livello dell’RNA. Infatti, in assenza di Npl3, abbiamo dimostrato la presenza di molecole di RNA di EXO1 non correttamente terminate. Questi dati, oltre al fatto che l’overespressione di EXO1 sopprime parzialmente il difetto di resection di cellule npl3Δ, suggeriscono che Npl3 partecipi alla regolazione della resection promuovendo la corretta biogenesi dell’mRNA di EXO1.
Riguardo al secondo progetto, Sae2 promuove l’attività endonucleasica di MRX durante la resection e regola negativamente il checkpoint Tel1-dipendente. Infatti, Sae2 limita l’accumulo di MRX alla lesione, riducendo sia il reclutamento che l’attività di segnalazione di Tel1. Non è ancora chiaro come le funzioni di Sae2 nel promuovere la resistenza ai danni e nell’inibire il checkpoint siano collegate. Tramite screening genetico, abbiamo identificato il mutante sae2-ms che, come accade in assenza di Sae2, iperattiva il checkpoint Tel1-dipendente, aumentando il reclutamento ai DSB sia di MRX che di Tel1. A differenza della delezione di Sae2, Sae2-ms non causa difetti di resection né di tethering, e non provoca sensibilità agli agenti genotossici. Inoltre, Sae2-ms provoca iperattivazione di Tel1, ma non di Rad53. Infatti, l’assenza di Sae2, ma non la presenza di Sae2-ms, aumenta l’interazione tra Rad53 e Rad9. Questi dati dimostrano che Sae2 regola il checkpoint sia controllando la rimozione di MRX dai DSB che limitando l’interazione Rad53-Rad9, e che l’inibizione di Rad53 è la principale responsabile della resistenza ai danni promossa da Sae2.Genomic integrity is threatened by DNA damage that, if not properly repaired, can be converted into mutations, whose accumulation leads to genomic instability, one of the hallmarks of cancer. Eukaryotic cells deal with DNA damage by activating DNA damage response. DNA double strand breaks (DSBs) are among the most dangerous DNA lesions. In Saccharomyces cerevisiae, DSBs are mainly repaired by Homologous Recombination (HR), which exploits a homologous sequence as a template to repair the damage. HR requires the DSB ends to be nucleolytically degraded in order to generate single-strand DNA (ssDNA) tails, in a process known as DSB end resection. Resection initiates with an endonucleolytic cleavage by the MRX complex together with Sae2, while resection extension is carried out by the nucleases Exo1 and Dna2. DNA damage checkpoint is a signal transduction cascade that halts the cell cycle in order to give cells sufficient time to repair the damage. In S. cerevisiae, DNA damage checkpoint is activated by the kinases Tel1 and Mec1, orthologues of human ATM and ATR. Once activated, Mec1 and Tel1 phosphorylate different substrates including the adaptor Rad9 and the effector kinase Rad53, which allow signal amplification. Both DNA end resection and DNA damage checkpoint must be finely regulated to ensure efficient DSB repair, avoiding excessive ssDNA generation, and to properly coordinate repair with cell cycle progression. In this PhD thesis, we provide evidences of a new level of resection regulation, based on the modulation of Exo1 amount by the RNA-binding protein (RBP) Npl3. We have also studied the role of Sae2 in DNA damage repair and checkpoint activation. Npl3 is a S. cerevisiae RBP, which plays a central role in RNA metabolism and is highly conserved from yeast to humans. Since emerging evidences support strong connections between RNA metabolism and genome integrity, we investigated if Npl3 was involved in DSB response. We demonstrated that the absence of Npl3 impairs the generation of long ssDNA tails at DSB ends. In particular, Npl3 promotes resection extension by acting in the same pathway of Exo1. Moreover, both the lack of Npl3 and the inactivation of its RNA-binding domains cause the reduction of Exo1 protein level. So, Npl3 promotes resection extension by regulating EXO1 at the RNA level. Indeed, we proved that the decrease of Exo1 level is due to the presence of not properly terminated EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, suggest that Npl3 participates in DSB end resection regulation by promoting the proper biogenesis of EXO1 mRNA. Concerning the second PhD project, Sae2 promotes MRX endonucleolytic activity during resection and negatively regulates Tel1-dependent checkpoint response. Indeed, Sae2 limits MRX accumulation at the damage site, thus reducing Tel1 recruitment and its signalling activity. How Sae2 functions in supporting DNA damage resistance and in inhibiting the DNA damage checkpoint are connected is still unclear. From a genetic screen, we identified the sae2-ms mutant that, similarly to Sae2 absence, upregulates Tel1 signalling activity by increasing both MRX and Tel1 recruitment to the DSBs. However, unlike SAE2 deletion, Sae2-ms does not cause any resection or tethering defect, nor any sensitivity to genotoxic agents. Moreover, Sae2-ms induces Tel1 but not Rad53 hyperactivation. Indeed Sae2 absence, but not Sae2-ms presence, increases Rad53-Rad9 interaction. These data indicate that Sae2 regulates checkpoint activation both by controlling MRX removal from the DSBs and by limiting Rad53-Rad9 interaction and that Rad53 downregulation is the main responsible for Sae2-promoted DNA damage resistance. Altogether, our results allow to better understand the molecular mechanisms involved in the control of DNA damage response processes.
7
Yin, Ling. "Activation of DNA Replication Initiation Checkpoint in Fission Yeast." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/194.
Full textAbstract:
In the fission yeast, Schizosacchromyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both stabilize stalled replication forks and to prevent premature entry into mitosis. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, my study shows that mutants defective in DNA replication initiation require the Chk1 kinase, rather than Cds1. This suggests that failed initiation events can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. I refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). The data shows that Chk1 is active in rid mutants when grown under semi-permissive conditions, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, an adaptor protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation. Like Mrc1, Swi1 and Swi3 have been hypothesized as a part of the replication fork protection complex (RFPC). They are required for maintaining the viability of rid mutants, but are not essential for activation of Chk1 in response to failed initiation events. This suggests that Mrc1 in conjunction with Swi1 and Swi3 function in a similar pathway to alleviate replicative stress resulting from defects in DNA replication initiation. Using flow cytometry, I demonstrate that inhibition of DNA replication initiation has no significant impact on the duration of S phase, suggesting dormant origins might be activated in response to defects in DNA replication initiation. Fission yeast Rad22 is implicated in forming nuclear foci in response to damaged DNA. By tracking YFP-labeled Rad22, I screened for potential DNA damage in rid mutants grown at semi-permissive temperatures, and the results show that DNA damage occurs as the result of defects in DNA replication initiation. I also identified camptothecin, a DNA topoisomerase I inhibitor that can at low dose (2 µM) induce the rid phenotype, suggesting our assay (Chk1-dependent, Cds1-independent) can be used to screen small molecule inhibitors that interfere with the initiation step of DNA replication.
8
Chahwan, Richard. "Analysis of the DNA damage checkpoint and of the cytokinesis machinery." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613310.
Full text
9
Choi, Jun-Hyuk Sancar Aziz. "Reconstitution of a human ATR-mediated DNA damage checkpoint prespone [sic]." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2460.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics." Discipline: Biochemistry and Biophysics; Department/School: Medicine.
10
Martinho, Rui Goncalo V. R. C. "Analysis of Rad3 and Chk1 checkpoint protein kinases." Thesis, University of Sussex, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297946.
Full text
11
Ponte, de Albuquerque Claudio. "A proteomics approach to study the DNA damage checkpoint in Saccharomyces cerevisiae." Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/ucsd/fullcit?p3398617.
Full textAbstract:
Thesis (Ph. D.)--University of California, San Diego, 2010.Title from first page of PDF file (viewed May 6, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 97-109).
12
Willis, Nicholas Adrian. "Checkpoint Regulation of Replication Forks in Response to DNA Damage: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/427.
Full textAbstract:
Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired.
Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial.
Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing.
Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V.
We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-xhypomorph to completely fail to slow.
We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were.
Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing.
Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.
13
Fletcher, Jessica Frances. "Novel variants of the DNA damage checkpoint protein Cds1 in Schizosaccharomyces pombe." Thesis, Bangor University, 2017. https://research.bangor.ac.uk/portal/en/theses/novel-variants-of-the-dna-damage-checkpoint-protein-cds1-in-schizosaccharomyces-pombe(9df1851b-ca3f-449f-880f-c8eb627cb786).html.
Full textAbstract:
In the model organism Schizosaccharomyces pombe, the Cds1 (checking DNA synthesis 1) kinase is activated at the S-phase checkpoint upon stalling of the replication fork during DNA synthesis. Under normal conditions (300C), the role of the full-length protein kinase is to activate downstream processes resulting in mitotic arrest,protection of the stalled replication fork, and prevention of continued DNA replication in an unfavourable environment. In this way, Cds1 acts to ensure the reversible arrest of DNA synthesis. However, under stress conditions such as raised temperature or specific DNA damaging drugs, shorter variants of this protein kinase are rapidly expressed. Initial experimentation revealed the origin of the predominant variant, named Cds1-B, as the internal translation initiation site Methionine 159. The expressed protein is N-terminally truncated, missing amino acids residues 1-158, and therefore lacking the regulatory SQ/TQ and FHA domains whilst retaining the kinase domain. Absence of these regulatory regions suggests that this variant is free to act outside of the chromatin environment to fulfil roles different to that of its full-length counterpart, however it is unlikely to be active as a kinase as it is unable to participate in the model of activation currently proposed in the literature. Experimentation in this project was aimed at elucidating the role of this Cds1-B variant through analysis of drug sensitivity and checkpoint control efficacy, and evaluation of kinase activity. Current literature and results discussed here suggest an interesting hypothesis in which variants are expressed in response to specific genotoxic stresses in order to self-regulate kinase activity and selectively mediate cellular response either by the DNA replication checkpoint effector Cds1 or the DNA damage checkpoint effector Chk1. Mediation of effector kinase action is a promising avenue for cancer treatment, and with a potential Cds1-B homologue for human Chk2 identified (splice variant isoform 13), gaining a greater nderstanding of these variant mechanisms in yeast will aid the development of new therapeutic interventions.
14
Can, Geylani. "S-phase checkpoint activity and function throughout the cell cycle." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/268506.
Full textAbstract:
DNA damage or replication stress during S-phase can activate the S-phase checkpoint which executes a variety of responses, such as the inhibition of origin firing and replication fork stabilisation. Deregulation of the S-phase checkpoint leads to genomic instability, which has been implicated in diseases such as cancer. In this thesis, I aimed to address whether the S-phase checkpoint is regulated outside of S-phase, and how the S-phase checkpoint targets its substrates in budding yeast. Although this checkpoint has thus far been associated exclusively with S-phase, it remains unknown whether its responses such as inhibition of origin firing can also occur in other phases of the cell cycle. To investigate this, the targets of the S-phase checkpoint for the inhibition of origin firing were analysed outside of S-phase upon DNA damage. Interestingly, I showed that the S-phase checkpoint effector kinase Rad53 phosphorylates its targets to inhibit origin firing outside of S-phase upon DNA damage when there is no replication. I then set out to test whether inhibition of origin firing by Rad53 outside of S-phase might be important for faithful DNA replication. Having shown that the checkpoint response is not specific for any cell cycle phases, I then tested how the specificity of Rad53 for its substrates might be determined. After demonstrating that the essential replication protein Cdc45 is required for Rad53 to phosphorylate the initiation factor Sld3, the key residues of Cdc45 necessary for Rad53 interaction were identified. A Cdc45 allele was produced by mutating the identified residues. This allele of Cdc45 is a separation-of-function mutant which prevents Sld3 phosphorylation upon DNA damage, but retains its function in DNA replication. Because Cdc45 travels with the replication fork, it is possible that Cdc45 also targets Rad53 to the replication fork to stabilise it upon replication stress. Overall, this thesis provides evidence that the S-phase checkpoint can function throughout the cell cycle and that Cdc45 targets Rad53 to some of its substrates, and possibly plays a role in replication fork stabilisation.
15
Francis, Kyle Evan. "Characterisation of checkpoint kinase 1 and 2 in ovarian cancer." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25956.
Full textAbstract:
CHEK1 inhibitors are currently in clinical trials for their ability to abrogate chemotherapy-induced CHEK1 activation and S phase arrest resulting in cancer cell apoptosis. No studies have yet identified ovarian cancers that could benefit from CHEK1-targeting therapy. I hypothesised that knowledge of CHEK1 and CHEK2 signalling in the DNA damage response can assist in identifying potential biomarkers for platinum responsiveness and CHEK-targeting therapy in ovarian cancer. In vitro studies investigated the CHEK1/2 inhibitor AZD7762 (AZD) and cisplatin (CP) in same patient-derived platinum-sensitive/resistant high-grade serous ovarian cancer cell lines (PEO1/PEO4 and PEO14/PEO23). Cytotoxicity assays confirmed higher CP IC50’s for PEO4 and PEO23 relative to PEO1 and PEO14 cell lines, respectively. AZD was more toxic to PEO1 cells and an additive effect of AZD with CP relative to CP alone was seen. A nontoxic AZD treatment to PEO4 cells sensitised the cells to CP when applied in combination. PEO14 and PEO23 cells had similar cytotoxicity profiles for combination treatments. BRDU DNA synthesis assays and cell cycle analysis revealed increased BRDU incorporation and accumulation in S phase when all cell lines were treated with CP. AZD treatment had a similar effect in PEO14 and PEO23 cells and increased the sub-G1 population, a marker of apoptotic DNA fragmentation, relative to control. Drug combination had no major effect on cell cycle distributions of both PEO14 and PEO23 cells relative to single agents but resulted in BRDU incorporation levels below CP and control levels for PEO14 cells. In PEO1 and PEO4 cells, AZD did not affect the cell cycle or DNA synthesis levels relative to control. Drug combination did not alter the cell cycle relative to CP treatment for PEO1 cells but decreased S phase and increased G2/M and sub-G1 populations in PEO4 cells. This was coupled with a decrease of CP-induced BRDU levels in PEO4 control levels. Apoptotic PARP cleavage/total PARP occurred early in CP treated PEO1 and PEO14 cells. A surrogate CHEK1/2 activity marker, p-CDC2 (Y15), decreased in all lines treated with AZD relative to control. Within PEO1 and PEO4 cells, greatest PARP cleavage was observed with combination treatment and coincided with high p-H2AX (S139), a DNA damage marker. p-CHEK1 (S317) and p-CHEK2 (T68), both ATR and ATM phosphorylation sites during DNA damage, increased for lone drug treatment and, to a greater extent, the combination drug treatments. PARP cleavage occurs across all treatments in PEO1 cells while it only occurs in the combination treatment for PEO4 cells. The latter coincides with a decrease in p-CHEK1 (S296) a CHEK1 autophosphorylation site, p-TP53 (S15), and p-BRCA1 (S1524), a homologous recombination marker, relative to the CP treated sample. In PEO14 and PEO23 cells, lone AZD and combination treatments had similar cleaved PARP/total PARP levels compared to the PEO14 CP treated cells. This was coupled with increased p-H2AX (S139), decreased CHEK1, and decreased CHEK2 autophosphorylation p-CHEK2 (S516). A human ovarian cancer xenograft model identified increases in p-H2AX (S139), CHEK1, p-CHEK1 (S317), p-CHEK2 (T68), and p-BRCA1 (S1524) in the carboplatin responsive cancers. In the paired pre- and post-chemotherapy human ovarian cancer samples, p-CHEK1 (S317) was elevated in post-chemotherapy responsive samples. In the first cohort, high p-CHEK1 (S317) was an independent poor overall survival biomarker and correlated with high p-H2AX (S139), MYC, p-CHEK1 (S296), p-CHEK2 (T68), p-CHEK2 (S516), and p-TP53 (S15). p-CHEK1 (S317) was associated with poor overall survival in serous ovarian cancers within the second pre-treatment ovarian cancer cohort. In conclusion, AZD can induce apoptosis in CP resistant cancer cells by synergising with CP to abrogate the S phase checkpoint, increase DNA damage, and inhibit CHEK1, and BRCA1 function. As a single agent, AZD can induce apoptosis by decreasing CHEK1 levels and CHEK2 activity. p- CHEK1 (S317) is a platinum responsive / poor prognostic biomarker.
16
Van, Der Sar Sjaak. "Proteomics of spindle checkpoint complexes and characterisation of novel interactors." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/11677.
Full textAbstract:
The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’. During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation of spindle checkpoint complexes signals the presence of incorrect spindle microtubule attachments to kinetochores. These complexes cooperate to suppress the activity of the anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these distinct complexes and analysing their composition by mass-spectrometry (MS) this work revealed several intriguing disparities between the two yeast species, and the way in which the Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’, which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate with Mad3 to form the ‘BUB+ spindle checkpoint complex’. In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19), Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants. Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA damage checkpoint or DNA replication checkpoint functions. Further examination revealed that Bub1 functions in the repair of DNA double strand breaks. Taken together, this work demonstrates that even though the molecular components of the spindle checkpoint pathway are conserved, their regulatory connections have to some extent diverged through molecular evolution. This process not only rewired, but entwined two molecular processes that together safeguard the genetic heritage of cells.
17
Bonilla, Carla Yaneth. "Co-localization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324582.
Full text
18
Ivanova, Tsvetomira Georgieva 1978. "The DNA damage and the DNA synthesis checkpoints converge at the MBF transcription factor." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/116932.
Full textAbstract:
DNA damage is an ongoing threat to both the ability of the cell to faithfully transmit genetic information to its offspring as well as to its own survival. In order to maintain genomic integrity, eukaryotes have developed a highly conserved mechanism to detect, signal and repair damage in DNA, known as the DNA damage response (DDR). In fission yeast the two DDR pathways converge at the regulation of single transcriptional factor complex (MBF) resulting in opposite directions. We have shown that when the DNA-synthesis checkpoint is activated, Max1 is phosphorylated by Cds1 resulting in the abrogation of its binding to MBF. As a consequence, MBF-dependent transcription is maintained active until cells are able to overcome the replication challenge. In contrast, upon DNA damage, Chk1 the effector kinase of DNA damage checkpoint is activated and blocks the cell cycle progression, inducing DNA repair and repressing the MBF dependent transcription. We have revealed that Cdc10 is the target of the DNA-damage checkpoint and when cells are treated with MMS or are exposed to IR, Chk1 phosphorylates Cdc10 inducing the exit of MBF from chromatin. The consequence is that under these conditions, MBF-dependent transcription is repressed. Thus, Max1 and Cdc10 couple normal cell cycle regulation and the DNA-synthesis and DNA-damage checkpoints into MBF.
19
Jones, Matthew Dunford. "Effects of radiation on the Gâ†2/M checkpoint in human tumour cells of differing radiosensitivities." Thesis, University of Liverpool, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387452.
Full text
20
Alpi, Arno. "DNA damage checkpoint pathways and the maintenance of genome stability in C. elegans." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-24487.
Full text
21
Volkmer, Elias. "Human checkpoint proteins hRad9, hHus1, and hRad1 form a DNA damage-responsive complex." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-27572.
Full text
22
Taschner, M. J. "Transcription-coupled nucleotide excision repair and its regulation by the DNA damage checkpoint." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18946/.
Full textAbstract:
Elaborate DNA repair mechanisms have evolved, allowing cells to repair damages in their genomes. Nucleotide excision repair (NER) removes a variety of helix-distorting lesions, including those caused by ultraviolet (UV) irradiation. NER operates via two subpathways. Transcription-coupled repair (TC-NER) rapidly removes transcription-blocking lesions in the transcribed strand (TS) of active genes, and in the yeast Saccharomyces cerevisiae depends on the factors Rad26 and Rpb9. Lesions in untranscribed DNA, including the non-transcribed strand (NTS) of active genes are removed slower by global genome repair (GG-NER). Besides activating specific DNA repair systems, DNA damage also leads to a global cellular response, known as the DNA damage checkpoint (DDC). Cell-cycle progression is temporarily stopped after DNA damage to allow sufficient time for repair and prevent replication or segregation of damaged chromosomes. The DDC is a complex signal transduction cascade involving a number of protein kinases, the central players in budding yeast being Mec1 and Tel1, the homologues of human ATR and ATM, respectively. Besides inhibiting cell-cycle progression, accumulating evidence suggests that DNA repair systems are also influenced by the checkpoint. I have investigated the rates of repair of UV lesions in checkpoint deficient strains of Saccharomyces cerevisiae and found that NER is significantly inhibited on both strands of an active gene in the absence of Mec1. The effect on NTS repair seems to be caused by deficient de novo synthesis of repair factors, whereas TC-NER is influenced mainly by post-translational modification of one or more pre-existing proteins. I have characterised a checkpoint-dependent phosphorylation of Rad26, and have shown using point mutants that this phosphorylation increases the TC-NER capacity of cells, establishing a new link between NER and the checkpoint. In addition to these results about the interplay between the DDC and NER pathways, preliminary data from two unrelated projects will be presented. One was an attempt to establish a system for analysis of NER factor recruitment to an artificial, highly UV-damage-prone DNA sequence. The other focussed on the regulation of UV-induced degradation of Rpb1, the largest RNA Polymerase II (RNAPII) subunit, by the DDC.
23
Ferrari, M. "CHARACTERIZATION OF FACTORS INVOLVED IN DNA DAMAGE CHECKPOINT RECOVERY AND ADAPTATION IN YEAST." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/229588.
Full textAbstract:
Genome maintenance and stability are essential goals for all the organisms in order to transfer the correct genetic information to the progeny and to keep fully functional the cellular metabolism. In eukaryotic cells, the presence of DNA lesions causes the activation of an evolutionary conserved mechanism called the DNA damage checkpoint that arrests the cell cycle and stimulates the repair pathways. Double strand breaks (DSBs) are deleterious lesions that can be a serious threat for the cell. In fact, the formation of only one DSB is enough to activate a robust checkpoint response. This DNA lesion is processed by several factors leading to the checkpoint factors recruitment and to the homologous recombination repair. After lesion repair the checkpoint is switched off through a process called recovery; however it has been demonstrated that damaged cells are able to inactivate the checkpoint and restart the cell cycle also in the presence of a persistent DNA lesion, through a checkpoint adaptation process. The reason why this process occurs is not understood, but it has been related to the unrestrained proliferation of cancer cell. In my laboratory we are interested in shedding light on the molecular mechanism of these checkpoint inactivation processes and in the characterization of the involved factors.
During the PhD I focused on the characterization of the functions and regulation of some factors already known to play a role in DSB ends processing and checkpoint switch off: the polo kinase Cdc5, the DNA translocase Tid1/Rdh54 and the nuclease-associated protein Sae2.
First of all we found that high levels of Cdc5 lead to checkpoint switch off and cell cycle re-enter. Relying on this data we decide to perform a biochemical screening in order to identify the Cdc5 targets in presence of DNA damage. This biochemical screening was based on a GST pulldown approach, coupled with tandem mass spectrometry protein identification. As expected, we identify many interactors and among them we found the repair protein Sae2. Interestingly, we found that in presence of elevated levels of Cdc5, Sae2 is hyperphospholylated and binds strongly to the DSB ends. In order to understand the functional role of the Cdc5-Sae2 interaction, I mutagenized different putative Cdc5 binding sites in Sae2. It turned out that Cdc5 binds a C-terminal region of Sae2, which is conserved in other eukaryotes orthologs.
The obtained Sae2 mutants give us interesting results that can be useful for the proper comprehension of the Sae2 function in DNA damage response. Indeed in this thesis I will present preliminary results on the characterization of the Sae2 role in the recovery process.
I was also involved in a project with the aim to study the regulation of Tid1/Rdh54 in the presence of DSB. Tid1 belongs to the Swi2/Snf2 family of chromatin remodellers, is an ATP-dependent DNA translocase able to induce DNA structure remodelling, Rad51 removal from double strand DNA and promote D-loop formation during homologous recombination. Moreover this protein has also a puzzling function in checkpoint inactivation during adaptation since TID1 deletion causes a permanent G2/M block in the presence of one irreparable DSB. I found that Mec1 and Rad53 checkpoint kinases, through a process that requires also the recombination factor Rad51, phosphorylate Tid1 in the presence of DSBs. I also found that Tid1 is recruited on to the DSB site, and that its ATPase activity is dispensable both for the loading and the phosphorylation of the protein. We believe that Tid1 phosphorylation is important to stabilize the binding of the protein on the lesion and to regulate its functional role during checkpoint adaptation.
24
DONDI, AMBRA. "ADAPTATION TO THE DNA DAMAGE CHECKPOINT REQUIRES THE REWIRING OF THE CELL CYCLE MACHINERY." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/609526.
Full textAbstract:
The DNA damage checkpoint is a surveillance mechanism evolved to preserve genome integrity in response to DNA damaging agents. The DNA damage checkpoint senses DNA insults and halts the cell cycle providing time and conditions to repair the lesion(s). If the damage is successfully repaired, cells reenter in the cell cycle in a process known as recovery to the DNA damage checkpoint. If the damage is not repaired, cells either undergo programmed cell death or override the checkpoint reentering the cell cycle in the presence of the lesion. This process, known as adaptation to the DNA damage checkpoint, represents an opportunity for cells to repair the damage in the following cell cycle. However, adaptation to the DNA damage checkpoint can be an unsafe event as daughter cells can accumulate genomic aberrations, therefore promoting genomic instability, and, indeed checkpoint adaptation has been described to occur also in cancer cells. Therefore, understanding the molecular mechanisms that drive checkpoint adaptation is a fundamental question to be addressed.
The molecular mechanism causing adaptation, as well as the players involved in this process, remains largely unknown. In budding yeast S. cerevisiae, the existence of a crosstalk between the cell cycle machinery and the DNA damage checkpoint have been suggested by two observations. First, the DNA damage checkpoint acts to halt cell cycle progression by directly inhibiting the pathways that control the exit from mitosis, namely the Cdc fourteen early anaphase release (FEAR) network and the mitotic exit network (MEN). Second, the activity of the FEAR network is required for checkpoint adaptation. Indeed, impairing the functions of single components of the FEAR network, namely Cdc5, Spo12 and Slk19, results in cells impaired in the adaptation process. While the molecular events for the DNA damage checkpoint activation have been intensely studied and
17
relatively well characterized, the molecular events that drive cell cycle resumption after checkpoint adaptation are less well understood. In the work presented in this thesis, we used and integrated different approaches, including genetics, single cell analyses, and fluorescence microscopy techniques to tackle this question.
Our findings indicate that the FEAR mutants (with the exception of Cdc5) are proficient in switching off the checkpoint but cannot exit mitosis, and suggest a more complex picture. As impairing the activity of single FEAR components does not affect exit from mitosis both in unperturbed conditions, and following checkpoint recovery, our studies unveil checkpoint adaptation as the rewiring of a cell cycle with peculiar features. From our investigations, we expect to elucidate the molecular circuitry underlying the rewiring of the cell cycle in persistent DNA damage conditions.
25
Cheung, Hiu-wing. "Significance of mitotic checkpoint regulatory proteins in chemosensitivity of nasopharyngeal carcinoma cells." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36438376.
Full text
26
MacDougall, Christina A. "Defining the structural determinants required for activation of the atr-dependent DNA damage checkpoint /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
Full text
27
Lopergolo, A. "CHK2 PHOSPHORYLATION OF SURVIVIN-DEX3 CONTRIBUTES TO A DNA DAMAGE-SENSING CHECKPOINT IN CANCER." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/171952.
Full textAbstract:
Survivin is a pivotal cancer gene with multiple roles in cell viability and mitosis, but the function(s) of its alternatively spliced isoforms has remained elusive. Here, we show that a survivin spliced variant that lacks exon 3 and contains a unique -COOH terminus sequence,
survivin-DEx3, is differentially expressed in cancer, compared to normal tissues, and correlates with aggressive disease and markers of unfavorable prognosis, by genome-wide bioinformatics analysis. Unlike other survivin variants, survivin-DEx3 localizes exclusively to nuclei in tumor cells, and is phosphorylated by the DNA damage checkpoint kinase, Chk2, on residues located in its unique -COOH terminus. Ala mutagenesis of the Chk2 phosphorylation sites prolongs survivin-DEx3 stability in tumor cells, inhibits the expression of Ser139 phosphorylated H2AX in response to double strand DNA breaks, and impairs colony formation in soft agar after DNA damage. Active Chk2 was detected at the earliest stages of the colorectal adenoma-to-carcinoma transition, persisted in advanced tumors, and correlated with increased survivin expression, in vivo. These data suggest that Chk2 phosphorylation of survivin-DEx3 contributes to a DNA damage-sensing checkpoint in tumor cells, which may affect sensitivity to genotoxic therapies.
28
Kermi, Chames. "Interaction fontionnelle entre le système de tolérance des lésions et le checkpoint des dommages à l'ADN : conséquences sur la stabilité du génome et l'oncogenèse." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONT3520/document.
Full textAbstract:
Notre génome subit constamment les effets néfastes des agents endommageant de l'ADN. Afin de se protéger de ces effets délétères, les cellules disposent d’un système de détection des dommages à l’ADN (point de contrôle ou « checkpoint »). Certaines lésions peuvent persister quand les cellules entrent en phase S et inhiber ainsi la synthèse de l’ADN en interférant avec les ADN polymérases réplicatives. Ceci peut provoquer des arrêts prolongés des fourches de réplication ce qui fragilise l’ADN. Pour préserver l’intégrité de l’information génétique, les cellules ont développé une voie de tolérance qui implique des ADN polymérases spécialisées dans la réplication des lésions, appelées ADN Polymérases translésionnelles (Pols TLS). Dans ce processus, PCNA joue le rôle de facteur d’échafaudage pour de nombreuses protéines impliquées dans le métabolisme de l'ADN. Les mécanismes de régulation des échanges entre les différents partenaires de PCNA ne sont pas très bien compris. Parmi les protéines qui interagissent avec PCNA, CDT1, p21 ou encore PR-Set7/Set8 sont caractérisées par une forte affinité pour cette protéine. Ces dernières possèdent un motif d’interaction particulier avec PCNA, nommé « PIP degron », qui favorise leur protéolyse d'une manière dépendante de l’E3 ubiquitine ligase CRL4Cdt2. Après irradiation aux UV-C, le facteur d’initiation de la réplication CDT1 est rapidement détruit d’une manière dépendante de son PIP degron, Dans la première partie de mon travail, j’ai contribué à comprendre le rôle fonctionnel de cette dégradation. Les résultats obtenus ont fourni des évidences expérimentales qui montrent que l’inhibition de la dégradation de CDT1 par CRL4Cdt2 dans les cellules de mammifères compromet la relocalisation des TLS Pol eta et Pol kappaen foyers nucléaires induits par les irradiations UV-C. On a constaté que seules les protéines qui contiennent un PIP degron interfèrent avec la formation de foyers de Pol eta. La mutagenèse du PIP degron de CDT1 a révélé qu'un résidu de thréonine conservé parmi les PIP degrons est essentiel pour l'inhibition de la formation des foyers des TLS Polymérases. Les résultats obtenus suggèrent que l’élimination de protéines contenant des PIP degrons par la voie CRL4Cdt2 régule le recrutement de TLS Polymérases au niveau des sites des dommages induits par les UV-C.Dans un second temps, on s’est intéressé à l’étude du checkpoint des dommages à l’ADN au cours de l’embryogénèse. En effet, dans les embryons précoces, le checkpoint est silencieux jusqu'à la transition de mid-blastula (MBT), en raison de facteurs maternels limitants. Dans ce travail, nous avons montré, aussi bien in vitro qu’in vivo, que l’ubiquitine ligase de type E3 RAD18, un régulateur majeur de la translésion, est un facteur limitant pour l’activation du checkpoint dans les embryons de xénope. Nous avons montré que l'inactivation de la fonction de l’ubiquitine ligase RAD18 conduit à l'activation du checkpoint par un mécanisme qui implique l’arrêt des fourches de réplication en face des lésions produites par les UV-C. De plus, nous avons montré que l'abondance de RAD18 et de PCNA monoubiquitiné (PCNAmUb) est régulée au cours de l’embryogénèse. À l’approche de la MBT, l’abondance de l'ADN limite la disponibilité de RAD18, réduisant ainsi la quantité de PCNAmUb et induisant la dé-répression du checkpoint. En outre, nous avons montré que cette régulation embryonnaire peut être réactivée dans les cellules somatiques de mammifères par l'expression ectopique de RAD18, conférant une résistance aux agents qui causent des dommages à l'ADN. Enfin, nous avons trouvé que l'expression de RAD18 est élevée dans les cellules souches cancéreuses de glioblastome hautement résistantes aux dommages de l'ADN. En somme, ces données proposent RAD18 comme un facteur embryonnaire critique qui inhibe le point de contrôle des dommages de l’ADN et suggèrent que le dérèglement de l’expression de RAD18 peut avoir un potentiel oncogénique inattenduOur genome is continuously exposed to DNA damaging agents. In order to preserve the integrity of their genome, cells have evolved a DNA damage signalling pathway known as checkpoint. Some lesions may persist when cells enter the S-phase and halt the progression of replicative DNA polymerases. This can cause prolonged replication forks stalling which threaten the stability of the genome. To preserve the integrity of genetic information, cells have developed a tolerance pathway which involves specialized DNA polymerases, called translesion DNA polymerases (TLS Pols). These polymerases have the unique ability to accommodate the damaged bases thanks to their catalytic site. In this process, PCNA acts as a scaffold for many proteins involved in DNA metabolism. The mechanisms governing the exchanges between different PCNA partners are not well understood. Among the proteins that interact with PCNA, CDT1, p21 and PR-Set7/set8 are characterized by a high binding affinity. These proteins have a particular interaction domain with PCNA, called "PIP degron", which promotes their proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. After UV-C irradiation, the replication initiation factor CDT1 is rapidly degraded in a PIP degron-dependent manner. During the first part of my work, we wanted to understand the functional role of this degradation. Our results have shown that inhibition of CDT1 degradation by CRL4Cdt2 in mammalian cells, compromises the relocalisation of TLS Pol eta and Pol kappato nuclear foci after UV-C irradiation. We also found that only the proteins which contain a PIP degron interfere with the formation of Pol eta foci. Mutagenesis experiments on CDT1 PIP degron revealed that a threonine residue conserved among PIP degrons is essential for inhibiting foci formation of at least two TLS polymerases. This results suggest that CRL4Cdt2-dependent degradation of proteins containing PIP degrons regulates the recruitment of TLS polymerases at sites of UV-induced DNA damage.During the second part of my thesis, we studied DNA damage checkpoint regulation during embryogenesis. Indeed, in early embryos, the DNA damage checkpoint is silent until the mid-blastula transition (MBT) due to maternal inhibiting factors. In this work, we have shown, both in vitro and in vivo, that the E3 ubiquitin ligase RAD18, a major regulator of translesion DNA synthesis, is a limiting factor for the checkpoint activation in Xenopus embryos. We have also shown that RAD18 depletion leads to the activation of DNA damage checkpoints by inducing replication fork uncoupling in front of the lesions. Furthermore, we showed that the abundance of RAD18 and PCNA monoubiquitination (PCNAmUb) is regulated during embryonic development. Near the MBT, the increased abundance of DNA limits the availability of RAD18, thereby reducing the amount of PCNAmUb and inducing the de-repression of the checkpoint. Moreover, we have shown that this embryonic-like regulation can be reactivated in somatic mammalian cells by ectopic expression of RAD18, conferring resistance to DNA damaging. Finally, we found high RAD18 levels in glioblastoma cancer stem cells highly resistant to DNA damage. All together, these data propose RAD18 as a critical factor that inhibits DNA damage checkpoint in early embryos and suggests that dysregulation of RAD18 expression may have an unexpected oncogenic potential
29
Putnam, Charles Wellington. "Integration of G2/M checkpoint, spindle assembly checkpoint,and Ran cycle regulators in the Saccharomyces cerevisiae DNA damage mitotic arrest response." Diss., The University of Arizona, 2004. http://hdl.handle.net/10150/280738.
Full textAbstract:
It is axiomatic that genomic stability is dependent upon regulatory pathways, termed checkpoints, which sense perturbations of cell cycle execution including damage to chromosomal DNA. In Saccharomyces cerevisiae, the principal DNA damage checkpoint is at G2/M. Heretofore, this and other checkpoints, such as the spindle assembly checkpoint, which is also operative at the metaphase/anaphase transition, have been viewed as essentially linear pathways, responding to a specific type of damage, signaling via sui generis proteins, and targeting a limited number of effectors for arrest. In a 1999 report, our laboratory reported bifurcation of the pathway downstream from Mec1 activation; this established the genetic basis of a previously unexplained phenotype: partial arrest defects of rad53 and pds1 strains. Moreover, the bifurcated pathway model established the framework for subsequent studies which determined the molecular targets of each. Here, I present evidence that the DNA damage and spindle checkpoint pathways are part of a network which is capable of bilaterally responding to damage. After DNA damage the Mec1-centric pathway is initially preeminent; the spindle pathway is redundant. After prolonged damage, however, the spindle checkpoint components become required for arrest. In studies of overexpression of the Mec1 homologue Tel1, I delineated the pathway responsible for the resultant constitutive delay; strikingly, the spindle components Mad1 and Mad2 are activated, not from the kinetochore, but from the nuclear periphery. This off-kinetochore pool of Mad proteins, anchored by the myosin-like proteins, Mlp1 and Mlp2, is likewise activated by the DNA damage response. Tel1 physically interacts with Xrs2 of the Mre11·Rad50·Xrs2 complex; evidence that Xrs2 participates in these same responses is also presented. Finally, the sensitivity of xrs2 to a microtubule poison, benomyl, suggests that M R·X may also participate in sensing spindle disruption. From a screen for novel checkpoint genes, I isolated Gtr1 (and later, Gtr2), which are negative regulators of the Ran cycle. Here, I provide evidence that deletion of either produces an identical partial arrest defect, which is independent of the Mec1-centric pathway. Because Gtr2 physically interacts with Esp1, I surmise that Gtr1/Gtr2 may enforce cytosolic localization of Pds1/Esp1 after DNA damage.
30
Amaral, Nuno. "The Aurora B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/403606.
Full textAbstract:
La coordinació de la citocinesis amb la segregació cromosòmica és essencial per mantenir l’estabilitat cromosòmica durant la proliferació cel•lular. Tant en llevat com en cèl•lules animals, els ponts de cromatina en anafase indueixen un retard en l’absició promogut pel “NoCut checkpoint” dependent d’Aurora B. No obstant això, es desconeix si la inhibició de l’abscisió protegeix del dany derivat dels ponts de cromatina i com aquests ponts son detectats. Hem descobert que els ponts de cromatina induïts per estrès replicatiu de l’ADN, defectes en condensació o en la topoisomerasa II atracen l’abscisió per mitjà del “NoCut checkpoint”. Aquest retard prevé el dany al DNA citocinesis-dependent i promou la viabilitat cel•lular, després de l’estrès replicatiu. Sorprenentment, el ponts de cromatina dels cromosomes dicèntrics no són suficients per desencadenar un “NoCut checkpoint”. A més a més, hem vist que l’estabilització del fus mitòtic en anafase, a través de APC-Cdh1, és essencial per la resposta NoCut i pot activar el NoCut en cèl•lules amb ponts dicentrics. Proposem que defectes estructurals dels cromosomes deguts a estrès replicatiu, descondensació o catenacions persistents, activen el NoCut a través d’una deficiència en l’activitat de la APC-Cdh1. Això estabilitza el fus mitòtic i permet a l’aurora B present a la “midzone” detectar els ponts de cromatina i inhibir l’abscisió.Coordination of cytokinesis with chromosome segregation is essential to maintain genome stability during cell proliferation. In yeast and animal cells, anaphase chromatin bridges induce an abscission delay through the Aurora B-dependent NoCut checkpoint. However, it is not known whether inhibition of abscission prevents damage of chromatin bridges and how these bridges are detected. We find that chromatin bridges induced by DNA replication stress or by defects in condensin or topoisomerase II delay abscission through the NoCut checkpoint. This delay prevents cytokinesis-dependent DNA damage and promotes cellular viability, after replication stress. Surprisingly, chromatin bridges from dicentric chromosomes are not sufficient to trigger NoCut. Additionally, we find that anaphase spindle stabilization, through APC-Cdh1, is essential for the NoCut response and can trigger NoCut in cells with dicentric bridges. We propose that chromosomal structural defects, from replication stress, decondensation or persistent catenations, trigger NoCut through impairment of APC-Cdh1 activity. This stabilizes the mitotic spindle and allows midzone-bound Aurora B to detect chromatin bridges and inhibit abscission.
31
Poitelea, Marius Ionica. "Study on the Rad3-Rad26 DNA damage checkpoint protein complex in fission yeast, Schizosaccharomyces pombe." Thesis, University of Sussex, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412675.
Full text
32
Porter-Goff, Mary Elizabeth. "The Role of the MRN Complex in the S-Phase DNA Damage Checkpoint: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/405.
Full textAbstract:
The main focus of my work has been the role of the MRN in the S-phase DNA damage checkpoint. The MRN plays many roles in cellular metabolism; some are checkpoint dependent and some are checkpoint independent. The multiple roles in cellular metabolism complicate study of the role of the MRN in the checkpoint. MRN mutations in budding yeast and mammals may display separation of function. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5’ resection to create single stranded DNA that is required for both signaling and homologous recombination. However, it is unclear if resection is essential for all of the cellular functions of MRN. Therefore I have made mutations to mimic those in budding yeast and mammals. I found that several alleles of rad32, as well as ctp1Δ, are defective in double-strand break repair and most other functions of the complex but maintain an intact S-phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads me to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.
One of the potential roles of Rad32 and the rest of the MRN complex is in sister chromatid exchange. The genetic requirements of sister chromatid exchange have been examined using unequal sister chromatid assays which only are able to assay exchanges that are illegitimate and produce changes in the genome. Most sister chromatid exchange must be equal to maintain genomic integrity and thus far there is no good assay for equal sister chromatid exchange. Yeast cells expressing the human equilibrative nucleoside transporter 1 (hENT1) and the herpes simplex virus thymidine kinase (tk) are able to incorporate exogenous thymidine into their DNA. This strain makes it possible for the fission yeast DNA to be labeled with halogenated thymidine analogs. This strain is being used to design an assay that will label one sister with BrdU and then DNA combing will be used to see equal sister chromatid exchange.
33
Al-Mahmoud, Widad Abdulsamad Mansour. "Novel variants of the DNA damage checkpoint protein Hus1 in fission yeast and human cells." Thesis, Bangor University, 2014. https://research.bangor.ac.uk/portal/en/theses/novel-variants-of-the-dna-damage-checkpoint-protein-hus1-in-fission-yeast-and-human-cells(dee8a56b-687f-4f24-9c6d-f81d73edc877).html.
Full text
34
CESENA, DANIELE. "The RNA processing proteins Xrn1 and Rrp6 regulate DNA damage checkpoint activation and telomere metabolism." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/158272.
Full textAbstract:
L’instabilità genomica è una delle caratteristiche principali delle cellule tumorali e può essere causata da difetti nella riparazione del DNA, dal mancato arresto del ciclo cellulare e dalla perdita della protezione telomerica all’estremità dei cromosomi, che porta alla degradazione e alla fusione delle estremità. Tra i vari tipi di danno al DNA, le rotture della doppia elica del DNA (Double-Strand Break o DSB) rappresentano una delle lesioni più pericolose, poiché possono causare mutazioni o riarrangiamenti cromosomici. In presenza di DSBs, le cellule eucariotiche attivano un checkpoint, dipendente dalle protein chinasi Tel1/ATM e Mec1/ATR, che arresta il ciclo cellulare finché il danno non è stato riparato. Mec1/ATR è attivata dal DNA a singolo filamento (ssDNA) ricoperto da RPA che si forma dopo il processamento nucleolitico (resection) delle estremità del DSB.
Una simile risposta è attivata anche quando le estremità naturali dei cromosomi eucariotici perdono la loro protezione, generando delle estremità simili ad un DSB che vengono riconosciute dal checkpoint e dai meccanismi di riparazione. Questa protezione è fornita da complessi nucleoproteici specializzati, chiamati telomeri. Il DNA telomerico è costituito da sequenze ripetute ricche in G che terminano con una coda a singolo filamento sporgente in 3’ (detta coda G), la quale è importante per l’estensione dei telomeri ad opera della telomerasi. Diverse proteine, tra cui il complesso CST, sono necessarie al mantenimento della struttura e della lunghezza dei telomeri sia in lievito che nei mammiferi.
Recenti dati sperimentali indicano che i fattori che processano l’RNA hanno un ruolo fondamentale nella stabilità del genoma e nel metabolismo telomerico, anche se il meccanismo è ancora poco compreso. In questa tesi abbiamo dimostrato che in Saccharomyces cerevisiae le proteine che degradano l’RNA Xrn1, Rrp6 e Trf4 promuovono l’attivazione di Mec1/ATR facilitando la formazione di DNA a singolo filamento ricoperto da RPA ai DSB. Inoltre, Xrn1 e Rrp6 sono necessarie per attivare il checkpoint anche ai telomeri deprotetti a causa del malfunzionamento di Cdc13, una delle subunità del complesso CST coinvolto nella protezione dei telomeri. Xrn1 facilita la formazione di DNA a singolo filamento ai DSBs promuovendo il caricamento del complesso MRX, mentre come svolga questa funzione ai telomeri deprotetti rimane ancora da chiarire. Al contrario, la generazione di ssDNA ai DSBs non è influenzata dalla mancanza di Rrp6 o Trf4, ma la loro assenza ostacola il reclutamento di RPA e quindi di Mec1 al sito di danno. L’inattivazione di Rrp6 e Trf4 non influenza né l’associazione di Rad51/Rad52 ai DSB né la riparazione della rottura attraverso la ricombinazione omologa (Homologous Recombination o HR), suggerendo che la piena attivazione di Mec1 richieda più DNA a singolo filamento ricoperto da RPA di quanto ne sia richiesto per la riparazione attraverso la ricombinazione omologa. Infine, Xrn1, regolando negativamente il trascritto di RIF1, è coinvolto nel mantenimento della lunghezza dei telomeri promuovendo l’associazione di Cdc13 indipendentemente dalla formazione di DNA a singolo filamento.
In conclusione, i nostri risultati forniscono un nuovo collegamento tra il processamento dell’RNA e il mantenimento della stabilità del genoma.Genome instability is one of the most pervasive characteristics of cancer cells. It can be due to DNA repair defects, failure to arrest the cell cycle and loss of telomere-end protection that lead to end-to-end fusion and degradation. Among the many types of DNA damage, the DNA Double Strand Break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Eukaryotic cells respond to DSBs by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR, in order to arrest the cell cycle until DSBs are repaired. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA) that arises upon nucleolytic degradation (resection) of the DSB. A similar checkpoint response is triggered when the natural ends of eukaryotic chromosomes lose their protection, resembling and being recognized as DSBs. This protection is provided by specialized nucleoprotein complexes called telomeres. Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3’-ended single-stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidences indicate that RNA processing proteins play critical, yet poorly understood, roles in genomic stability and telomere metabolism. We provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate activation of Mec1/ATR by promoting the generation of RPA-coated ssDNA at intrachromosomal DSBs. Xrn1 and Rrp6 are also required to activate a Mec1/ATR-dependent checkpoint at uncapped telomeres due to loss of the CST component Cdc13. Xrn1 promotes checkpoint activation by facilitating the generation of ssDNA at both DSBs and uncapped telomeres. Xrn1 exerts this function at DSBs by promoting the loading of the MRX complex, whereas how it does at uncapped telomeres remains to be determined. By contrast, DSB resection is not affected by the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Finally, we demonstrate that Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the RIF1 transcript. Our results provide novel links between RNA processing and genome stability.
35
Li, Zhengke. "New Insights into the Roles of Human DNA Damage Checkpoint Protein ATR in the Regulation of Nucleotide Excision Repair and DNA Damage-Induced Cell Death." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etd/1782.
Full textAbstract:
Integrity of the human genome is frequently threatened by endogenous and exogenous DNA damaging reagents that may lead to genome instability and cancer. Cells have evolved multiple mechanisms to repair DNA damage or to eliminate the damaged cells beyond repair and to prevent diverse diseases. Among these are ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage checkpoint and nucleotide excision repair (NER) that are the major pathways by which cells handle ultraviolet C (UV-C)- or other exogenous genotoxin-induced bulky DNA damage. However, it is unclear how these 2 pathways may be coordinated. In this study we show that ATR physically interacts with NER factor xeroderma pigmentosum group A (XPA) where an ATR phosphorylation site on serine 196 is located. Phosphorylation of XPA on serine 196 is required for repair of UV-induced DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the UV-induced XPA phosphorylation and DNA repair. Moreover, we show that depletion of p53, a downstream checkpoint of ATR, and inhibition of p53 transcriptional activities reduced the UV-induced XPA import. Furthermore, we found that the ATR-directed XPA nuclear import happens primarily in the S phase of the cell cycle. In effort to determine the mechanism involved in the XPA nuclear import, we found that, in addition to the nuclear localization signal (NLS) of XPA, importin-α4 is required for the UV-induced XPA nuclear import in an ATR-dependent manner. These data suggest that NER could be regulated by the ATR-dependent checkpoint via modulation of XPA phosphorylation and nuclear import. In a separate study we show that, upon UV damage, cytoplasmic ATR translocates to mitochondria, blocks the recruitment of proapoptotic Bcl-2–associated X (Bax) protein to mitochondria and prevents the loss of mitochondrial membrane potential (ΔΨ) and apoptosis. Bax-depletion reduces the effect of ATR on ΔΨ. Remarkably, the cytoplasmic ATR exhibits no checkpoint kinase activity, a hallmark function of nuclear ATR. Silencing of ATR’s kinase activity failed to affect Bax relocalization to mitochondria. These results reveal a novel checkpoint-independent antiapoptotic function of ATR at mitochondria in the cellular response to DNA damage.
36
Cheung, Hiu-wing, and 張曉穎. "Significance of mitotic checkpoint regulatory proteins in chemosensitivity of nasopharyngeal carcinoma cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B37233919.
Full text
37
Sawicka, Marta. "Dissecting the DNA damage response : structural and biochemical insights into the Mec1-Ddc2 checkpoint kinase complex." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/45289.
Full textAbstract:
The survival and proper functioning of an organism depends on the faithful replication of its genetic material. The yeast checkpoint kinase Mec1 and its integral partner Ddc2 (human ATR and ATRIP respectively) play a pivotal role in initiating checkpoint signalling as a response to DNA damage. Mec1 is activated by cell cycle speci c activators that act through conserved aromatic residues located in their unstructured C-terminal regions. Since the molecular details of the activation mechanism of Mec1 are not fully understood yet, we studied the structure of the Mec1-Ddc2 complex and its interactions with the activator Dpb11 protein. Our ndings provide the rst insights into the overall architecture of Mec1-Ddc2 and serve as a basis for a proposed framework for the activation of Mec1. Our single-particle cryo-electron microscopy reconstruction shows Mec1- Ddc2 exists as a dimer of heterodimers and associates through intertwining arm regions consisting of the N-terminal Mec1 HEAT repeats of the adjacent monomers. The Ddc2 subunit, which extends from these repeats, further stabilises the oligomerization interface. The head of the structure accommodates the kinase domain located at the conserved C-terminus of Mec1. Due to a head-to-head dimer conformation, the kinase domains face each other although they are fully separated indicating they do not cause structural impediments that would block substrate access. The interactions between the N-terminus of Ddc2 and the C-terminal tail of Dpb11 mediate the recruitment of the activator to the checkpoint complex. The Ddc2 subunit, which neighbours the kinase domain, not only provides structural support but also facilitates Mec1 activation by bringing Dpb11 into close proximity to the active site. We propose a model where the activator stimulates the kinase activity via multiple interactions between the Mec1 and Ddc2 subunits of the complex that trigger small allosteric changes within the kinase domains. We hope such insights will pave the way to a full mechanistic understanding of this important signalling pathway.
38
Sertic, S. "Human exonuclease 1 connects the ner response and the checkpoint activation after UV induced DNA damage." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/158338.
Full textAbstract:
Human Exo1 is required to activate the checkpoint response after UV irradiation in human cells. hExo col-localizes with NER factors at the sites of UV lesions and hEXO1 down-regulation by siRNA technology affects DNA repair synthesis.
39
Munoz, Marcia Medicine UNSW. "The EDD protein is a critical mediator in the DNA damage response." Awarded by:University of New South Wales. Medicine, 2006. http://handle.unsw.edu.au/1959.4/25977.
Full textAbstract:
An intact cellular response to DNA damage is important for the maintenance of genomic stability and tumour prevention. EDD, the human orthologue of Drosophila melanogaster ???hyperplastic discs???, is over-expressed or mutated in a number of common human cancers. EDD is a progestin regulated gene that encodes an E3 ubiquitin ligase involved in cell communication and cell adhesion, and although it has also been implicated in the DNA damage response through its association with DNA damage proteins, a definitive role has yet to be demonstrated. The work presented herein shows that EDD is necessary for an adequate cellular response to double-strand DNA breaks. Cells depleted of EDD exhibit reduced survival, radio-resistant DNA synthesis and failure to maintain G2/M arrest following DNA damage induced by phleomycin exposure. Furthermore, EDD-depleted cells display impaired activating phosphorylation and kinase activity of the checkpoint kinase CHK2 after DNA damage. These effects appear to be largely modulated through a phospho-dependent interaction involving the CHK2 FHA domain and a region of EDD spanning a number of putative FHA-binding threonines. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasise the potential importance of EDD in cancer.
40
Campbell, Callum James. "Time to quit? : non-genetic heterogeneity in cell fate propensity after DNA damage." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275600.
Full textAbstract:
Cellular checkpoints are typically considered to both facilitate the ordered execution of the cell cycle and to act as a barrier to oncogene driven cell cycles and the transmission of unresolved genetic lesions from one phase to the next. Furthermore, these mechanisms are also believed to underpin the responses of cells, both in normal and cancerous tissues, to those therapies that either directly or indirectly generate DNA damage. In recent studies however, it has become clear these checkpoints permit the passage of significant genomic aberrations into subsequent cell cycle phases and even descendant cells, and that heterogeneous responses are apparent amongst genetically identical cells. The consequences of this checkpoint ‘negligence’ remain relatively uncharacterised despite the importance of checkpoints in current models for how genomic instability is avoided in the face of ubiquitous DNA damage. Unresolved DNA damage is presumably inherited by subsequent cell cycle phases and descendant cells yet characterisation of the consequences of this has been relatively limited to date. I therefore utilised microscopy-based lineage tracing of cells expressing genetically encoded fluorescent sensors, particularly the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Sakaue-Sawano et al., 2008), with semi-automated image analysis to characterise the response of single cells and their descendants to DNA lesions across multiple cell cycle generations. This approach, complemented by generational tracing by flow cytometry, permitted me to characterise the timing of cell fate determination in treated and descendant cells, the non-genetic heterogeneity in checkpoint responses and overall lineage behaviour, correlations between cells (similarly to Sandler et al., 2015) and cell cycle timing dependencies in the response to DNA damaging agents. With these single cell analytical approaches I show that the consequences of DNA damage on descendant cell fate is dramatic, suggesting checkpoint mechanisms may have consequences and even cooperate across phases and generations. U2OS cell lineages traced for three generations following the induction of DNA damage in the form of strand breaks showed greatly induced cell death in the daughters and granddaughters of DNA damaged cells, termed delayed death. Furthermore, lineage behaviour was characterised as highly heterogeneous in when and whether cell death occurred. Complementary flow cytometric approaches validated the findings in U2OS cells and suggested HeLa cells may show similar behaviour. These findings indicate that checkpoint models need to incorporate multigenerational behaviour in order to better describe the response of cells to DNA damage. Understanding the processes governing cell fate determination in descendant cells will impact upon our understanding of the development of genomic instability during carcinogenesis and how DNA-damaging chemotherapeutics drive cells to ‘quit’ the cell cycle.
41
Körner, Cindy. "Development of bioreductive inhibitors of checkpoint kinase 1 to target hypoxic tumours." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:10928a98-bfc0-4628-8edc-295642d4c05c.
Full textAbstract:
Hypoxia (low physiological O2 levels) is a characteristic of solid tumours. It not only alters the chemical microenvironment of a tumour but initiates a number of mechanisms which enable cells to cope and thrive under these conditions, resulting in therapy-resistant and aggressive tumours. The replication stress induced by severe hypoxia activates a DNA damage response which involves the kinases ATR and Chk1. Moreover, periods of hypoxia are often followed by reoxygenation, which induces DNA damage. Chk1 inhibitors have been used to potentiate chemotherapy with cytotoxic agents and have recently been proposed as single agents in tumours with high levels of replication stress. However, inhibition of Chk1 also affects normal DNA replication, cell cycle progression and DNA repair. The herein presented study chose known inhibitors of Chk1 and, with methods of synthetic organic chemistry, modified them into agents to selectively target hypoxic cells. Three different Chk1 inhibitors were selected and bioreductive analogues synthesised which were evaluated in chemical, biochemical and cellular assays. We found a convenient route to access a precursor of the bioreductive 2-nitroimidazole group and established a three-step protocol for the testing of bioreductive drugs. This protocol allows us to determine whether a bioreductive drug undergoes reduction and prodrug activation. In addition, bioreductive Chk1 inhibitors were shown to induce DNA damage and cellular toxicity in a hypoxia-selective fashion. While reduction of the prodrugs occurred in all three cases, fragmentation was always the rate-limiting step. We propose that the use of bioreductive Chk1 inhibitors is a promising strategy to target the most therapy-resistant tumour fraction while sparing normal tissue.
42
Sommariva, Elena. "Role of fission yeast replication pausing and termination proteins in replication associated DNA damage checkpoint and repair." Thesis, St George's, University of London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418176.
Full text
43
Vidanes, Genevieve M. "Suppression of the DNA damage checkpoint by the Saccharomyces cerevisiae polo-like kinase, CDC5, to promote adaptation." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3352477.
Full text
44
Nyberg, Kara Ann. "Analysis of RAD9 functions: Roles in the checkpoint response, DNA damage processing, and prevention of genomic instability." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280312.
Full textAbstract:
In the 15 years since Rad9's discovery, we have come to understand a great about Rad9 biology, yet numerous questions still remain. Especially intriguing questions include: (a) How does Rad9 get localized and/or recognize DNA damage? (b) How does Rad9 activate downstream checkpoint proteins? and (c) Does Rad9 play additional roles in recognizing and/or repairing DNA damage that have yet to be discovered? In an effort to try to answer some of these questions, I analyzed the effects of various RAD9 mutations. To assess the contribution of RAD9 to inhibiting DNA degradation and its role in the cell cycle arrest and DNA damage repair responses, I performed a pentapeptide mutagenesis screen in order to obtain RAD9 separation-of-function mutants that were proficient for one known phenotype and deficient in the other. I was able to obtain 2 such mutants that were hypomorphic in their ability to prevent DNA degradation and completely proficient for arresting the cell cycle in the presence of DNA damage and for repairing such damage. Despite many efforts, I was unable to enhance the hypomorphic dysfuntion of these mutants in preventing DNA degradation such that a suppressor screen to identify other genes in this pathway could be performed. In another effort to try to understand the role of the BRCT domains for RAD9 functions, I analyzed the effects of other various RAD9 mutants. By deletion analysis, I was able to determine that the predominant function of RAD9's BRCT domains is to mediate concentration of the Rad9 protein for function by two means: (1) by conferring stability to the Rad9 protein, and (2) by homodimerizing Rad9 to increase its local concentration to enable interactions with downstream checkpoint components.
45
Rawal, C. "ROLE OF POLO KINASE CDC5 AND SLX4-RTT107 COMPLEX IN CHECKPOINT SIGNALING DURING DNA DAMAGE IN S. CEREVISIAE." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/335192.
Full textAbstract:
The integrity of genomic DNA is continuously jeopardized through of environmental stresses such as UV light, ionizing radiations and various chemicals in addition to cellular byproducts such as reactive oxygen species. Furthermore, structural or chemical hindrances also affect the basic cellular processes (replication, transcription and translation) compromising genome stability. All the eukaryotic cells have thus evolved mechanisms to detect such genomic lesions and activate a surveillance mechanisms termed as checkpoint activation to arrest cell cycle, which in term provide time to repair the lesion using a suitable pathway to maintain genome stability. The resumption of cell cycle after the repair is also an important and finely regulated mechanisms. Indeed, resumption of cell cycle in case of faulty/un-repaired damage compromises genome integrity and may lead to cancer.
In this thesis, I studied the role of Polo-kinase Cdc5 and DNA repair scaffold complex-Slx-Rtt107, specifically in response to one of the most deleterious lesion, DNA double strand break (DSB) in budding yeast Saccharomyces cerevisiae. The human counterpart Polo-like kinase 1 is overexpressed in many cancers, while Slx4/FANCP is one of the proteins involved in Fanconi anemia repair pathway.
In first part, we characterized the role of phosphorylation of Threonine 238 in the activation loop of the Cdc5 kinase domain in unperturbed cell cycle and in response to repairable and unrepairable DSB. Using alanine/ aspartic acid mutagenesis and genetic approaches we delineated the requirement of T238 phosphorylation of Cdc5. Interestingly, we discovered that absence of T238 phosphorylation of Cdc5, even though doesn’t affect the normal cell cycle, affects kinase activity and leads to defect in checkpoint adaptation and recovery after one DSB. Importantly, we also found that cdc5-T238A cells also have altered genome stability, assessed by using multiple genetic approaches.
In second part, we characterized the role of Slx4-Rtt107 complex in modulating the level of checkpoint signalling and initial processing of DSB. Indeed in the absence of functional Slx4-Rtt107 complex, we found slower processing of DSB and hyper-activated checkpoint signalling which is due to increased binding of checkpoint adaptor protein Rad9 at the lesion. Importantly, this hyper-activated checkpoint has consequent effect on cell cycle resumption and proliferation in response to various DNA damaging agents.
46
Shirazi, Fard Shahrzad. "The Heterogenic Final Cell Cycle of Retinal Horizontal Cells." Doctoral thesis, Uppsala universitet, Medicinsk utvecklingsbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-222559.
Full textAbstract:
The cell cycle is a highly complex process that is under the control of several pathways. Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.
47
TROVESI, CAMILLA. "Regulation of the DNA damage response by cyclin-dependent kinase in "Saccharomyces cerevisiae ”." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/41816.
Full textAbstract:
The eukaryotic cell division cycle comprises a series of events, whose ordering and correct progression depends on the oscillating activity of cyclin-dependent kinases (Cdks), which safeguard timely duplication and segregation of the genome. Since genome integrity is constantly threatened by both endogenous and exogenous sources of DNA damage, cell division cycle is intimately connected to an evolutionarily conserved DNA damage response (DDR) in order to guarantee the faithful transmission of genetic information from one cell to its daughter and to ensure cell survival. In fact, the DDR involves DNA repair pathways that reverse DNA lesions, as well as DNA damage checkpoint pathways that inhibit cell-cycle progression while repair occurs. The DNA damage checkpoint is activated in the presence of DNA damage or replicative stress and is based on signal transduction cascades of protein kinases that recognize damaged DNA, transduce and amplify the damage signal, and target several effector proteins to prevent cell cycle progression and to couple it with the DNA repair capacity. In addition to driving cell-cycle arrest, these pathways control the activation of DNA repair pathways, the proper completion of DNA replication, the activation of transcriptional programs and, in some cases, the commitment to cell death by apoptosis.
There is increasing evidence that Cdks are not only downstream targets of the DDR, but also participate in the DDR regulation, both by leading to a strong checkpoint activation and by promoting DNA repair by homologous recombination (HR). The most dangerous DNA lesions are the DNA double-strand breaks (DSBs) that can be repaired either by non-homologous end joining (NHEJ) or by HR. While NHEJ directly relegates the broken DNA ends, HR uses homologous DNA sequences as a template to form recombinants that are either crossover or noncrossover with regard to flanking parental sequences. Furthermore, a DSB flanked by direct DNA repeats can be repaired by a particular HR pathway called single-strand annealing (SSA), which results in DSB repair with concomitant deletion of one repeat and of the intervening sequence. All HR processes initiate with extensive 5’ to 3’ end-processing (a process referred to as 5’-3’ resection) of the broken ends to yield 3’-ended single-stranded DNA (ssDNA) tails, which are bound by Replication Protein A (RPA). RPA is then displaced by Rad51 to form nucleoprotein filaments that can catalyse homologous pairing and strand invasion.
The choice between NHEJ and HR pathways is tightly regulated during the cell cycle and HR is generally restricted to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. This cell cycle specificity depends on Cdk (Cdk1 in Saccharomyces cerevisiae) activity, which initiates HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps was unknown. To address this question, we explored the Cdk1 requirement in the execution of different HR processes in S. cerevisiae. In order to bypass the Cdk1 requirement for resection we analyzed cells lacking Yku heterodimer and/or the checkpoint protein Rad9, which are known as negative regulators of DSB resection. We showed that yku70Δ cells, which accumulate ssDNA at the DSB ends independently of Cdk1 activity, are able to repair a DSB by SSA in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by SSA and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes. As crossovers during mitotic cell growth have the potential for deleterious genome rearrangements when the sister chromatid is not used as repair template, this additional function of Cdk1 in promoting crossovers can provide another safety mechanism to ensure genome stability.
During DNA replication cells are particularly sensitive to DNA damage. Eukaryotic cells respond to replication interference through a complex signal-transduction pathway, known as the S-phase checkpoint, whose key players in S. cerevisiae are the Mec1 and Rad53 kinases. Both Mec1 and Rad53 are essential for budding yeast cell viability and mec1 and rad53 checkpoint mutants are extremely sensitive to agents that cause replicative stress, such as hydroxyurea (HU) and methyl methanesulfonate (MMS). The sensor kinase Mec1 is recruited to stalled replication forks, where it activates the effector kinase Rad53. The activation of both these kinases maintain the integrity/activity of the replication forks, stimulate deoxyribonucleotides (dNTPs) production, inhibit the firing of late replication origins and prevent accumulation of aberrant DNA structures. A fundamental question to be addressed was which of the above checkpoint-regulated process(es) is/are critical for the maintenance of cell viability. We investigated this question searching for extragenic mutations suppressing the hypersensitivity to HU of mec1 mutant. By characterizing one of the identified suppressor mutations, we provide evidence that decreased activity of Cdk1 alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G1 and mitosis. In fact, delaying either the G1/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to HU and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions.
In conclusion, our results suggest that Cdk1 influences the DDR through multiple mechanisms. Indeed, Cdk1 is required for DSB-induced checkpoint activation, DSB repair by homologous recombination, and crossover formation. On the other hand, Cdk1 activity must be carefully regulated, because too much Cdk1 activity can affect genome integrity, at least when the checkpoint is not functional.
48
CASARI, ERIKA. "Resection of DNA double-strand breaks: novel regulatory mechanisms by checkpoint proteins and chromatin remodelers." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/365496.
Full textAbstract:
L'instabilità genomica è una delle caratteristiche delle cellule tumorali e può essere dovuta a difetti nella riparazione dei danni al DNA. Tra le differenti tipologie di danno al DNA, le rotture della doppia elica di DNA sono lesioni citotossiche che devono essere riparate per garantire stabilità genomica ed evitare la morte cellulare. Le cellule eucariotiche affrontano questi danni attivando una risposta con differenti vie molecolari. Tra esse il meccanismo NHEJ lega direttamente le estremità rotte del DNA. Oppure il meccanismo HR utilizza il cromatidio fratello/il cromosoma omologo come templato per riparare la rottura. HR è avviata dalla degradazione nucleolitica (resection) delle estremità 5' del DSB. La resection è un processo a due fasi: la prima fase, short-range, è catalizzata dal complesso MRX/MRN che, insieme a Sae2/CtIP catalizza un taglio endonucleolitico alle estremità 5’ del DNA rotto. Dopo di che, la seconda fase, long-range, prevede l’intervento delle nucleasi Exo1 e Dna2/Sgs1. Esse sono necessarie per generare code di 3’ssDNA. In seguito a una rottura della doppia elica di DNA, le cellule attivano anche un’altra via chiamata checkpoint da danno al DNA, che coordina la riparazione del danno con la progressione del ciclo cellulare. Tra i principali attori del checkpoint ci sono le chinasi Mec1/ATR e Tel1/ATM. Tel1 riconosce le rotture del doppio filamento di DNA non processate, mentre Mec1 è attivato dal DNA a singolo filamento, prodotto dal processo di resection. Una volta stimolate, queste due chinasi apicali attivano per fosforilazione le chinasi effettrici Rad53/CHK2 e Chk1/CHK1. Questa attivazione richiede anche la proteina conservata Rad9/53BP1 la cui associazione al DNA coinvolge diverse vie. Resta da determinare come la short-range resection sia regolata e contribuisca all'attivazione del checkpoint. In questa tesi, ho contribuito a dimostrare che l’inibizione della long-range resection induce una risposta di checkpoint che dipende dal complesso di checkpoint 9-1-1, che recluta Rad9 al DNA danneggiato. Inoltre, il complesso 9-1-1, indipendentemente da Rad9, limita la short-range resection regolando negativamente la nucleasi di MRX. La riparazione dei danni della doppia elica di DNA coinvolge anche la cromatina. Infatti, i genomi eucariotici sono compattati in una struttura cromatinica che limita l'accesso al DNA agli enzimi dedicati alla riparazione dei danni e solleva la questione di come avvenga la resection in tale contesto. È noto che la cromatina che affianca una rottura della doppia elica di DNA subisce ampie modificazioni da parte di una serie di rimodellatori della cromatina. Pertanto, nella seconda parte di questa tesi, ho studiato il ruolo della proteina di rimodellamento della cromatina Dpb4 nella riparazione dei danni. Nel lievito gemmante, la proteina conservata Dpb4 presenta un dominio istonico ed è condivisa da due complessi proteici: il rimodellatore della cromatina ISW2 e l'oloenzima DNA Pol epsilon. In S. cerevisiae, Dpb4 interagisce con Dls1 nel complesso ISW2 e con Dpb3 nel complesso Pol epsilon. In questa tesi ho dimostrato che Dpb4 promuove la rimozione degli istoni e la resection interagendo con Dls1 per facilitare l'associazione dell'ATPasi Isw2 al DNA danneggiato. Inoltre, promuove l'attivazione del checkpoint interagendo con Dpb3 per facilitare l'associazione di Rad9. Nell'ultima parte della tesi, per comprendere meglio il legame tra il rimodellamento della cromatina e la resection dei danni al DNA, ho contribuito a studiare il ruolo del rimodellatore della cromatina Chd1, frequentemente mutato nel cancro alla prostata. Abbiamo dimostrato che Chd1 partecipa in entrambe le fasi di resection, promuovendo l'associazione di MRX ed Exo1 alle estremità di una rottura del DNA. Inoltre, Chd1 consente la rimozione degli istoni vicino alle estremità del DNA danneggiato promuovendone la riparazione con il meccanismo di HR.Genome instability is one of the hallmarks of cancer cells and can be due to DNA repair defects. Among different types of DNA damage, DNA DSBs are cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. Eukaryotic cells deal with DSBs by activating the DNA damage response, that comprises pathways devoted to repair DNA breaks. DSBs can be repaired by NHEJ, which directly ligates the broken DNA ends, or by HR, which uses sister chromatids/homologous chromosomes as a template to repair the DNA break. HR is initiated by nucleolytic degradation (resection) of the 5’-terminated strands at both DSB ends. DSB resection is a two-step process, in which an initial short-range step is catalyzed by Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that, together with Sae2 (CtIP in mammals), catalyzes an endonucleolytic cleavage of the 5’strands. Then, a long-range resection step is carried out by the nucleases Exo1 and Dna2/Sgs1 to generate long 3’ssDNA tails. The DDR comprises also surveillance mechanisms, called DNA damage checkpoint, that couple DSB repair with cell cycle progression. Major checkpoint players include the apical protein kinases Mec1/ATR and Tel1/ATM. Tel1 recognizes unprocessed DSBs, while Mec1 is activated by RPA-coated ssDNA that is generated during the resection process. Once activated, these protein kinases activate by phosphorylation the effector kinases Rad53/CHK2 and Chk1/CHK1. This activation requires the conserved adaptor protein Rad9/53BP1, whose association to chromatin involves multiple pathways. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. In this thesis, I contributed to show that abrogation of long-range resection induces a checkpoint response that depends on the checkpoint complex 9-1-1, which recruits Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated. Repair of DSBs occurs in a chromatin context. In fact, eukaryotic genomes are compacted into chromatin, which restricts the access to DNA of the enzymes devoted to repair DNA DSBs and raises the question as to how DNA end resection occurs in the context of chromatin. For this reason, chromatin near DSBs undergoes extensive modifications by a series of conserved chromatin remodelers that are recruited to DNA DSBs. Thus, given the importance of chromatin remodeling in DSB repair, in the second part of this thesis, I investigated the role of the chromatin remodeling protein Dpb4 in DSB repair. Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase epsilon holoenzyme. In S. cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol epsilon complex. I showed that Dpb4 plays two functions in sensing and processing DSBs. It promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. In the last part of my thesis, to better understand the link between chromatin remodeling and DNA end resection, I contributed to examine the role in DSB repair of the S. cerevisiae chromatin remodeler Chd1, whose human counterpart is frequently mutated in prostate cancer. We showed that Chd1 participates in both short- and long- range resection by promoting the association of MRX and Exo1 to the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity.
49
Di, Cicco Giulia [Verfasser], and Heinrich [Akademischer Betreuer] Leonhardt. "Cell cycle and DNA damage-dependent control of the checkpoint mediator Rad9 / Giulia Di Cicco ; Betreuer: Heinrich Leonhardt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/117587860X/34.
Full text
50
Wang, Jianwei [Verfasser]. "Batf defines a differentiation checkpoint limiting hematopoietic stem cell self renewal in response to DNA damage / Jianwei Wang." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2012. http://d-nb.info/1023728540/34.
Full text