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1
Atkinson, John David. "Regulation of the E. coli Replicative Helicase DnaB by the Helicase Loader DnaC." Thesis, University of Glasgow, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485809.
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The helicase proteins directly ~esponsible for unwinding chromosomal DNA during DNA replication in bacteria, archaea and eukaryotes adopt a ring-shaped conformation for rapid displacement ofthe parental DNA duplex. As the DNA substrate is engulfed by the helicase during t~slocation, accessory proteins are required for placement ofthe helicase onto the DNA substrate either by breaking the ringed complex, thus allowing DNA to pass into the central channel, or by assembling the helicase around the DNA. In E. coli, the replicative helicase is a hexameric complex of six DnaB monomers, whilst the accessory loading partner is DnaC. Six DnaCmonomers associate with DnaB6 to assist in the loading ofthe helicase onto the DNA substrate. However, once present on the DNA, the presence ofDnaC on the DNA-bound helicase is thought to prevent the initiation of translocation. Only when DnaC has dissociated from DnaB6 will the helicase be permitted to commence unwinding ofthe duplex DNA. ,. Using in vitro enzymatic assays to identify the helicase activity ofDnaB on partial duplex DNA substrates, I have shown that when the concentration ofDnaC exceeds that ofDnaB, translocation and strand displacement by the helicase is not hindered when only one DNA strand is incorporated. When the helicase translocates over two DNA strands, however, movement is terminated via interaction with DnaC. More specifically, DnaB translocation is halted when DnaC is coupled with ATP, while interaction with ADP permits continued helicase movement. In vivo, the presence of DnaC within the DNA replication machinery complex could prove advantageous to the cell, as chromosomal duplication will only be permitted at bonefide replication forks. Keywords: DNA replication, helicase, DnaB, DnaC
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Arribas, Bosacoma Raquel. "Resolució de l'estructura tridimensional de l'helicasa hexamètrica DnaB." Doctoral thesis, Universitat de Girona, 2009. http://hdl.handle.net/10803/7639.
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Es presenta el model atòmic a 4.5 Å de DnaB, la principal helicasa replicativa bacteriana, d'Aquifex aeolicus. És un anell hexamèric de 100 Å d'amplada i 80 Å d'alçada amb dues capes de simetria diferenciada, la dels dominis N-terminals en C3 i la dels C-terminals propera a C6. El diàmetre central és de 25 Å al llarg d'ambdues capes, principal diferència amb les estructures prèvies, on era 25 Å més estret a la capa N-terminal. L'estretament s'origina pel trencament d'una de les dues superfícies d'interacció entre monòmers N-terminals, cosa que augmenta la flexibilitat del subdomini implicat. Només l'ssDNA pot atravessar l'anell, quan a les estructures prèvies hi podia passar tant ssDNA com dsDNA. L'estructura aquí presentada és més propera a la conformació funcional de DnaB durant la realització de l'activitat helicasa, mentre que les anteriors correspondrien a la forma inactiva o a la conformació capaç de translocar-se sobre dsDNA.DnaB is the main replicative helicase in bacteria. An atomic model for the DnaB from Aquifex aeolicus at a 4.5 Å resolution is presented. It´s a ring-shaped homohexamer (100 Å width and 80 Å hight) with two simmetry layers, a C3 N-terminal layer and an almost C6 C-terminal one. The diameter of the central channel is 25 Å along both layers, being the main diference with the previously solved structures, which were 25 Å smaller along the N-terminal layer. This is due to one of the previous interacting surphaces being lost in the current structure, thus enabling a higher felxibility of the subdomain involved. Only ssDNA can pass trhough the ring, while both ssDNA and dsDNA could in the previous structures. So, the present structure is closer to the functional conformation, while the previous ones would correspond to the inactive form or the conformation that is only able to translocate along dsDNA.
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Weigelt, Johan. "Development of new NMR techniques and the structure of the N-terminal domain of Escherichia coli DnaB helicase /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3414-2.
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McRobbie, Anne-Marie M. "Splitting, joining and cutting : mechanistic studies of enzymes that manipulate DNA." Thesis, University of St Andrews, 2010. http://hdl.handle.net/10023/951.
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DNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea.
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Song, Daqing. "Homologous Strand Exchange and DNA Helicase Activities in Plant Mitochondria." Diss., CLICK HERE for online access, 2005. http://contentdm.lib.byu.edu/ETD/image/etd931.pdf.
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Leah, Labib. "Helicase Purification for DNA Sequencing." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31341.
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BACKGROUND: A method to increase accuracy and ease-of-use, while decreasing time and cost in deoxyribonucleic acid (DNA) sequence identification, is sought after. Helicase, which unwinds DNA, and avidin, which strongly attracts biotin for potential attraction of biotinylated DNA segments, were investigated for use in a novel DNA sequencing method.
AIM: This study aimed to (1) purify bacteriophage T7 gene product 4 helicase and helicase-avidin fusion protein in a bacterial host and (2) characterize their functionality.
METHODS: Helicase and helicase-avidin were cloned for purification from bacteria. Helicase-avidin was solubilised via urea denaturation/renaturation. DNA and biotin binding were assessed using Electrophoretic Mobility Shift Assays and biotinylated resins, respectively.
RESULTS: (1) Helicase and helicase-avidin proteins were successfully purified. (2) Helicase protein was able to bind DNA and avidin protein strongly bound biotin.
CONCLUSION: Helicase and helicase-avidin can be purified in a functional form from a bacterial host, thus supporting further investigation for DNA sequencing purposes.
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Rudolf, Jana. "Characterisation of XPD from Sulfolobus acidocaldarius : an iron-sulphur cluster containing DNA repair helicase." Thesis, St Andrews, 2007. http://hdl.handle.net/10023/159.
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Korhonen, Jenny. "Functional and structural characterization of the human mitochondrial helicase /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-102-2/.
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Johnson, Vinu. "Structural and Biophysical Studies of Single-Stranded DNA Binding Proteins and dnaB Helicases, Proteins Involved in DNA Replication and Repair." University of Toledo / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1198939056.
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Tasleem, Arsala. "Helicase Attachment to Carbon Nanotubes for DNA Sensor." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37392.
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Purpose: Current DNA detection techniques require complicated procedures, specialized training, expensive equipment, invasive samples and significant amount of sample collection and processing time. The purpose of this research was to develop a rapid, accurate, non-invasive and electronic method of DNA sensing that harnesses natural unwinding properties of DNA helicase by attaching it to Carbon Nanotubes.
Methods:
a. A literature review on methods of attaching proteins to carbon nanotubes was conducted
b. A design of the biosensor was developed based on previously reported attachment methods for other proteins
c. A part of the sensor was developed by attaching DNA helicase to carbon nanotubes
d. The result was tested for preservation of helicase functionality and carbon nanotube electronic structure integrity
Results:
a. Helicase was successfully attached to carbon nanotubes
b. Helicase was found to retain its NTP hydrolysis function, DNA binding and DNA unwinding ability upon attachment
c. Carbon nanotube electronic structure and function was not compromised upon attachment
Conclusions: Non-specific attachment of helicase to carbon nanotubes preserves enzyme structure and function, allowing rapid DNA unwinding at an in vitro rate comparable to DNA helicase.
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Dillingham, Mark Simon. "Biochemical studies on DNA helicases." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312245.
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Mankouri, Hocine William. "DNA helicases and yeast ageing." Thesis, University of Liverpool, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367550.
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Tognetti, Silvia. "Helicase activation mechanisms during initiation of DNA replication." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/40925.
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This thesis provides new insights into the recruitment mechanisms of factors to replicative origins that are important for helicase activation during initiation of DNA replication in budding yeast. DNA replication origins are recognised by the Origin Recognition Complex (ORC), which recruits, during late M-phase, Cdc6, Cdt1 and MCM2-7 to form a pre-replication complex (pre-RC). MCM2-7 represents the core of the eukaryotic replicative helicase, but it is inactive within the pre-RC. In early S-phase, the pre-RC is converted into the pre-initiation complex (pre-IC), leading to stable Cdc45/GINS/MCM2-7 (CMG) complex formation and helicase activation. This process depends on the recruitment of a series of factors (Sld3, Cdc45, Sld2, Sld7, Dpb11 ...) and is regulated by kinases (DDK and S-CDK) but their specific function and mechanism of action is largely unknown. A systematic pairwise interaction analysis was performed using purified and in vitro translated proteins from Saccharomyces cerevisiae in order to understand how these factors interact with each other and MCM2-7, allowing helicase loading and activation. During this study a new network of interactions was identified, which is required for the stable recruitment of Cdc45 to MCM2-7. This was concomitant with the in vitro reconstitution of Cdc45 loading with purified proteins, which described Sld2, in addition to Sld3, as Cdc45 loading factor. Moreover, the Mcm2 subunit of the MCM2-7 hexamer was identified as a central player in Cdc45 recruitment, representing a binding surface for Sld2, Sld3 and Cdc45. The interaction sites in these proteins were mapped and mutants were generated to disrupt the formation of the complex containing Cdc45. The results obtained in this work constitute a comprehensive overview on the interactions occurring during pre-RC, and CMG formation. Additionally, they provide important details on the mechanisms leading to helicase activation and contribute to the understanding of this still poorly defined step of DNA replication.
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Harrison, Ryan M. "Molecular biophysics of strong DNA bending and the RecQ DNA helicase." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f02fc167-b705-4275-a413-21d13b5d94c3.
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Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.
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Ozdemir, Ahmet Yunus. "BIOCHEMICAL STUDIES OF DNA POLYMERASE THETA." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/560412.
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Biomedical SciencesPh.D.
POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Pol theta in replication repair. DNA polymerase theta is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining of DNA double-strand breaks. How full-length human DNA polymerase theta performs microhomology-mediated end-joining and is regulated by the helicase and disordered central domain remains unknown. We find that the helicase upregulates DNA polymerase theta microhomology-mediated end-joining activity in an ATPase-independent manner. Using single-particle microscopy, we find that DNA polymerase theta forms large multimeric complexes that promote DNA accumulation and end-joining. We further find that the disordered central domain regulates DNA polymerase theta multimerization and governs its DNA substrate requirements for end-joining. In summary, these studies identify major regulatory functions for the helicase and central domains in DNA end-joining and the structural organization of DNA polymerase theta.
Temple University--Theses
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Cavanagh, David R. "DNA helicase II and exonuclease V of Escherichia coli." Thesis, University of Newcastle Upon Tyne, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293560.
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Huber, Michael D. "Structure-function analysis and substrate specific inhibition of RecQ helicases /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9253.
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McFarlane-Majeed, Laura. "A functional characterisation of the DNA helicase Ch1R1 in DNA replication and repair." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5919/.
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ChlR1 is a DNA helicase implicated in diverse cellular processes including sister chromatid cohesion and DNA replication and repair. However, the mechanism by which ChlR1 participates in these processes is unknown. Data presented in this thesis show that siRNA-mediated depletion of ChlR1 causes increased sensitivity to chemically-induced replication stress. Treatment of ChlR1-depleted cells with hydroxyurea results in increased mono-ubiquitination of PCNA and increased chromatin-associated RPA, indicating stalled DNA replication. Furthermore, ChlR1 is recruited to chromatin following hydroxyurea treatment, supporting a role in the stabilisation of forks during replication stress. Fibroblasts derived from a Warsaw Breakage Syndrome (WABS) patient caused by mutation of ChlR1 (G57R) have both defective sister chromatid cohesion and G2 checkpoint following radiation-induced damage. Complementation with wild-type ChlR1 rescued this mutant phenotype while a known helicase dead mutant of ChlR1 (K50R) or the WABS-associated mutants G57R or ΔK897 did not. However, increased and prolonged Chk1 activation was observed in both K50R and ΔK897 complemented cells after treatment with hydroxyurea while the G57R was comparable to wild-type. These data suggest that the novel WABS mutation (G57R) may retain some wild-type ChlR1 activity and offer important insight into the molecular basis of the WABS phenotype.
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Sedman, Tiina. "Characterization of the yeast Saccharomyces Cerevisiae mitochondrial DNA helicase hmi1 /." Online version, 2005. http://dspace.utlib.ee/dspace/bitstream/10062/1332/5/sedman.pdf.
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Levin, Mikhail Konstantinovich. "DNA UNWINDING MECHANISM OF THE HELICASE FROM HEPATITIS C VIRUS." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1017851412.
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Jordan, Christian. "Helicase-SSB Interactions In Recombination-Dependent DNA Repair and Replication." ScholarWorks @ UVM, 2014. http://scholarworks.uvm.edu/graddis/270.
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Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair.
Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda and Gp32 play a critical role in regulating replication fork restart during recombination repair. When the leading strand polymerase stalls at a site of ssDNA damage and the lagging strand machinery continues, Gp32 binds the resulting ssDNA gap ahead of the stalled leading strand polymerase. We found that a Gp32 cluster on leading strand ssDNA blocks Dda loading on the lagging strand ssDNA, blocks stimulation of fork progression by Dda, and stimulates Dda to displace the stalled polymerase and the 3' end of the daughter strand. This unwinding generates conditions necessary for polymerase template switching in order to regress the DNA damage-stalled replication fork. Helicase trafficking by Gp32 could play a role in preventing premature fork progression until the events required for error-free translesion DNA synthesis have taken place. Interestingly, we found that Dda helicase activity is strongly stimulated by the N-terminal deletion mutant Gp32-B, suggesting the N-terminal truncation to generate Gp32-B reveals a cryptic helicase stimulatory activity of Gp32 that may be revealed in the context of a moving polymerase, or through direct interactions of Gp32 with other replisome components.
Additionally, our findings support a role for Dda-Gp32 interactions in double strand break (DSB) repair by homology-directed repair (HDR), which relies on homologous recombination and the formation of a displacement loop (D-loop) that can initiate DNA synthesis. We examined the D-loop unwinding activity of Dda, Gp41, and UvsW, the D-loop strand extension activity of Gp43 polymerase, and the effect of the helicases and their modulators on D-loop extension. Dda and UvsW, but not Gp41, catalyze D-loop invading strand by DNA unwinding. The relationship between Dda and Gp43 was modulated by the presence of Gp32. Dda D-loop unwinding competes with D- loop extension by Gp43 only in the presence of Gp32, resulting in a decreased frequency of invading strand extension when all three proteins are present. These data suggest Dda functions as an antirecombinase and negatively regulates the replicative extension of D- loops. Invading strand extension is observed in the presence of Dda, indicating that invading strand extension and unwinding can occur in a coordinated manner. The result is a translocating D-loop, called bubble migration synthesis, a hallmark of break-induced repair (BIR) and synthesis dependent strand annealing (SDSA). Gp41 did not unwind D- loops studied and may serve as a secondary helicase loaded subsequent to D-loop processing by Dda. Dda is proposed to be a mixed function helicase that can work both as an antirecombinase and to promote recombination-dependent DNA synthesis, consistent with the notion that Dda stimulates branch migration. These results have implications on the repair of ssDNA damage, DSB repair, and replication fork regulation, which are highly conserved processes sustained in all organisms.
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Manhi, Hoida Ismail Abdel-Aziz. "Bloom DNA helicase facilitates homologous recombination between diverged homologous sequences." Kyoto University, 2011. http://hdl.handle.net/2433/142044.
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Zhong, Yichen. "Mechanistic Studies of Human Chromodomain-Helicase-DNA-Binding Protein 4." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/23473.
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In eukaryotic cells, genomic DNA is organized into small units called nucleosomes, each of which generally consists of a histone octamer and DNA wrapping around this histone core, so that the whole genome can be efficiently packed into a small nucleus. On the other hand, the presence of nucleosome remodelers ensures the genetic information still remains accessible to regulatory factors. Although it was known for a long time that these remodellers can weaken the interaction between the DNA and histones in an ATP-dependent manner to expose part of the DNA sequence, the underlying mechanisms have not been fully explored, especially for the CHD remodeler family. In the project, we assembled recombinant nucleosomes and used them as the substrate to test the remodelling ability of CHD4 and also NuRD (Nucleosome Remodelling Deacetylase) complex, using single molecule FRET (fluorescence resonance energy transfer) experiments. The FRET experiment involves fluorescent labelling of one of the histones and also of the DNA, which allows the monitoring of the translocation of the nucleosome in real time. Other experimental approaches, including real-time remodelling rate monitoring, and gel-based remodelling assays were also carried out. Together with structural studies, these experiments were used to delineate the biochemical mechanism of remodelling by CHD4-family chromatin remodelling proteins.
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Tokonzaba, Etienne. "Molecular mechanism of SV40 large tumor antigen helicase /." Connect to abstract via ProQuest. Full text is not available online, 2007.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 82-92; 128-134). Online version available via ProQuest Digital Dissertations.
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Ali, Yusuf I. "Design, synthesis and characterisation of tool inhibitors targeting BLM helicase." Thesis, University of Sussex, 2018. http://sro.sussex.ac.uk/id/eprint/80487/.
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Richards, Jodi Dominique. "Helicases and DNA dependent ATPases of Sulfolobus solfataricus /." St Andrews, 2008. http://hdl.handle.net/10023/474.
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Richards, Jodi D. "Helicases and DNA dependent ATPases of Sulfolobus solfataricus." Thesis, University of St Andrews, 2008. http://hdl.handle.net/10023/474.
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DNA is susceptible to various types of damage as a result of normal cellular metabolism or from environmental sources. In order to maintain genome stability a number of different, partially overlapping DNA repair pathways have evolved to tackle specific lesions or distortions in the DNA. Nucleotide excision repair (NER) is highly conserved throughout eukarya, bacteria and archaea and predominantly targets lesions that result from exposure to UV light, for example cyclobutane pyrimidine dimers and 6-4 photoproducts. The majority of archaea possess homologous of the eukaryotic repair genes and this thesis describes the isolation and the characterization of two XPB homologues identified in the crenarchaeon Sulfolobus solfataricus, SsoXPB1 and SsoXPB2. Human XPB is one of 10 proteins that make up the TFIIH transcription complex. The activity of XPB is tightly controlled by protein interactions, in particular with p52, which stimulates the ATPase activity of XPB. Rather than a conventional helicase, human XPB is thought to act as an ATP dependent conformational switch. Consistent with human XPB, however, the S. solfataricus proteins were unable to catalyse strand separation and the identification of an archaeal protein partner, Bax1, for SsoXPB2 was one of the focuses of this project. In order to maintain genome stability, the DNA must be replicated accurately with each cell cycle. When the advancing replication fork stalls at a lesion or a DNA break, it is crucial that the fork is reset and that replication continues to completion. The helicase Hel308 is thought to clear the lagging strand template of a stalled replication fork in order for replication restart to proceed via homologous recombination (HR). Although the specific function of Hel308 is not well understood, the possibilities are described in this thesis. Strand exchange proceeds to form a D-loop, followed by branch migration to increase regions of heterology during the synapsis stage of HR. No motors for branch migration have previously been recognised in archaea, although the identification of a possible candidate was investigated during this project.
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Ochem, Alexander. "Properties of two DNA helicases of human cells." Thesis, Open University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299015.
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Syed, Salahuddin. "Nonreplicative DNA Helicases Involved in Maintaining Genome Stability." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6408.
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Double-strand breaks and stalled forks arise when the replication machinery encounters damage from exogenous sources like DNA damaging agents or ionizing radiation, and require specific DNA helicases to resolve these structures. Sgs1 of Saccharomyces cerevisiae is a member of the RecQ family of DNA helicases and has a role in DNA repair and recombination. The RecQ family includes human genes BLM, WRN, RECQL4, RECQL1, and RECQL5. Mutations in BLM, WRN, and RECQL4 result in genetic disorders characterized by developmental abnormalities and a predisposition to cancer. All RecQ helicases have common features including a helicase domain, an RQC domain, and a HRDC domain. In order to elucidate the role of these domains and to identify additional regions in Sgs1 that are required for the maintenance of genome integrity, a series of systematic truncations to the C terminus of Sgs1 were created. We found that ablating the HRDC domain does not cause an increase in accumulating gross chromosomal rearrangements (GCRs). But deleting the RQC domain and leaving the helicase domain intact resulted in a rate similar to that of a helicase-defective mutant. Additionally, we exposed these truncation mutants to HU and MMS and demonstrated that losing up to 200 amino acids from the C terminus did not increase sensitivity to HU or MMS, whereas losing 300 amino acids or more led to sensitivity similar to that of an sgs1∆ cell. These results suggest that the RQC domain, believed to mediate protein-protein interactions and required for DNA recognition, is important for Sgs1’s role in suppressing GCRs and sensitivity to HU and MMS, whereas the HRDC domain that is important for DNA binding is not necessary. RecQL5 is a RecQ-like helicase that is distinct from the other members through its three different isoforms, RecQL5α, RecQL5β, and RecQL5ɣ. It has a helicase domain and an RQC domain, but lacks the HRDC domain that other RecQ-like helicases possess. In contrast to Blm, Wrn, and RecQL4, no human disorder has been associated with defects in RecQL5. For this reason the role of RecQL5 in the cell has remained largely unknown. To try to elucidate the pathways RecQL5 may be involved in we performed a yeast two hybrid to identify RecQL5-interacting proteins. We found that RecQL5 interacts with Hlp2, an ATP-dependent RNA helicase, and Ube2I, a SUMO-conjugating enzyme. These novel interactions shed light on a potential role of RecQL5 in the cell as a transcriptional regulator.
Saccharomyces cerevisiae, Rrm3, is a 5’-3’ DNA helicase that is part of the Pif1 family of DNA helicases and is conserved from yeast to humans. It was initially discovered as a suppressor of recombination between tandem arrays and ribosomal DNA (rDNA) repeats. In its absence there are increased rates of extra-chromosomal rDNA circles, and cells accumulate X-shaped intermediates at stalled forks. Rrm3 may be involved in displacing DNA-protein blocks and unwinding DNA to facilitate fork progression. We used stable isotope labeling by amino acids in cell culture (SILAC)- based quantitative mass spectrometry in order to determine proteins that deal with the stalled fork in the absence of Rrm3. We found that in the absence of Rrm3 and increased replication fork pausing, there is a requirement for the error-free DNA damage bypass factor Rad5 and the homologous recombination factor Rdh54 for fork recovery. We also report a novel role for Rrm3 in controlling DNA synthesis upon exposure to replication stress and that this requirement is due to interaction with Orc5, a subunit of the origin recognition complex. Interaction of Orc5 was found to be located within a 26-residue region in the unstructured N-terminal tail of Rrm3 and loss of this interaction resulted in lethality with cells devoid of the replication checkpoint mediator Mrc1, and DNA damage sensitivity with cells lacking Tof1. In this study we describe two independent roles of Rrm3, a helicase-dependent role that requires Rad5 and Rdh54 for fork recovery, and a helicase-independent role that requires Orc5 interaction to control DNA synthesis.
Our data provides novel insight into the role of DNA helicases and their role in protecting the genome. Through yeast genetics it was possible to determine the importance of the C terminus of Sgs1 and elucidate new RecQL5 interacting partners that shed light onto roles for RecQL5 distinct from other RecQ like helicases. Quantitative mass spectrometry allowed us to take on a more global view of the cell and determine how it responds to replication fork pausing in the absence of Rrm3. Using both proteomics and yeast genetics we were able to better understand how these DNA helicases contribute to maintaining genome stability.
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Chilton, Scott S. "A mutational analysis of the Bacillus subtilis competence helicase ComFA." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11560.
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Genetic competence is a developmental process in bacteria that allows natural transformation. Competent Gram positive bacteria such as Bacillus subtilis carry a cytosolic helicase which is required for efficient transformation. In this work ComFA is confirmed as a DEAD-box helicase. I also describe a new accessory motif in ComFA that contributes to transformation independently of the helicase activity in ComFA. The newly discovered metal-binding motif consists of four cysteines which are required for transformation and zinc binding. While the zinc finger is required for full function, it is not required for DNA binding. As DEAD-box family helicases are generally non-processive, it appears that at least part of the rapid DNA uptake process is mediated by a non-processive helicase. Active uptake using the ComFA helicase motor may be required to maintain the integrity of the incoming DNA to allow subsequent recombination.
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Markham, Jonathan Edward. "Biotin-containing enzymes from Brassica napus and Arabidopsis thaliana." Thesis, Durham University, 1996. http://etheses.dur.ac.uk/1648/.
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Cassidy, Sarah Anne. "Stabilisation of DNA triple helices." Thesis, University of Southampton, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242535.
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Novoa, Carolina. "RecQ-like helicase SGS1 counteracts DNA : RNA hybrid induced genome instability." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/60964.
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Dividing cells are constantly under threat from both endogenous and exogenous DNA damaging stresses that can lead to mutations and structural variations in DNA. One contributor to genome instability is three-stranded DNA:RNA hybrid structures called R-loops. Though R-loops are known to induce DNA damage and DNA replication stress, it is unclear whether they are recognized and processed by an established DNA repair pathway prior to inducing DNA breaks. Canonically, DNA repair proteins work downstream of R-loop-induced DNA damage to stimulate repair and suppress genome instability. Recently, the possibility that some DNA repair pathways actively destabilize R-loops, thus preventing unscheduled DNA damage has emerged. Here we identify the helicase SGS1 as a suppressor of R-loop stability. Our data reveals that SGS1 depleted cells accumulate R-loops. In addition, we define a role for transcription in genome instability of cells lacking SGS1, which is consistent with an R-loop based mechanism. Hyper-recombination in SGS1 mutants is dependent on transcript length, transcription rate, and active DNA replication. Also, rDNA instability in sgs1Δ can be suppressed by ectopic expression of RNaseH1, a protein that degrades DNA:RNA hybrids. Interestingly, R-loops are known to form at rDNA loci. We favour a model in which SGS1 contributes to the stabilization of stalled replication forks associated with transcription complexes, and unresolved DNA:RNA hybrids. Finally, we showed that knockdown of the human Sgs1 orthologue BLM in HCT116 cells also led to the accumulation of more R-loops than control HCT116 cells. In summary, our data supports the idea that some DNA repair proteins involved in replication fork stabilization might also prevent and process R-loops.Science, Faculty of
Graduate
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ROSSI, SILVIA EMMA. "INTERPLAY BETWEEN THE DNA HELICASES PIF1 AND RRM3, THE NUCLEASE DNA2 AND THE CHECKPOINT PATHWAYS IN THE MAINTENANCE OF THE DNA REPLICATION FORK INTEGRITY." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/471797.
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Eukaryotic cells have evolved the ATR/hCHK1, MEC1/RAD53 kinase-mediated signal transduction pathway, known as replication checkpoint, to protect and stabilize stalled replication forks in human cells and budding yeasts, respectively.
rad53 mutants, exposed to high doses of the DNA replication inhibitor hydroxyurea (HU), accumulate hemireplicated, gapped and reversed forks, while treatments with low HU doses induce massive chromosome fragmentation.
The aim of my work was to better understand the molecular mechanisms through which Rad53 prevents unusual alterations of the architecture of the stalled replication forks and chromosome fragility, under replication stress.
We revealed that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites in unperturbed conditions, are detrimental in rad53 mutants experiencing HU-induced replication stress. Rrm3 and Pif1 ablation synergistically rescues cell lethality, chromosome fragmentation, replisome dissociation, fork reversal and ssDNA gaps formation at the forks of rad53 cells exposed to replication stress. We provide evidence that Pif1 and Rrm3 associate with stalled DNA replication forks and are regulated through Rad53-mediated phosphorylation.
Our findings uncover a new replication-stress-induced regulative loop in which Rad53 down regulates the Pif1 DNA helicases at the stalled replication forks.
In the second part of this thesis we examined the crosstalk between Rrm3, Pif1, the mediator of the DNA damage checkpoint Rad9 and the nuclease Dna2, during unperturbed DNA replication. The experimental evidence collected in this second part of the project, together with pioneering work previously reported from other laboratories, strongly suggests that Dna2, Pif1 and Rrm3 cooperate to finalize late stages of DNA replication.
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Klaue, Daniel. "DNA Unwinding by Helicases Investigated on the Single Molecule Level." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-97596.
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Each organism has to maintain the integrity of its genetic code, which is stored in its DNA. This is achieved by strongly controlled and regulated cellular processes such as DNA replication, -repair and -recombination. An essential element of these processes is the unwinding of the duplex strands of the DNA helix. This biochemical reaction is catalyzed by helicases that use the energy of nucleoside triphophate (NTP) hydrolysis. Although all helicases comprise highly conserved domains in their amino acid sequence, they exhibit large variations regarding for example their structure, their function and their target nucleic acid structures. The main objective of this thesis is to obtain insight into the DNA unwinding mechanisms of three helicases from two different organisms. These helicase vary in their structures and are involved in different pathways of DNA metabolism. In particular the replicative, hexameric helicase Large Tumor-Antigen (T-Antigen) from Simian virus 40 and the DNA repair helicases RecQ2 and RecQ3 from Arabidopsis thaliana are studied. To observe DNA unwinding by these helicases in real-time on the single molecule level, a biophysical technique, called magnetic tweezers, was applied. This technique allows to stretch single DNA molecules attached to magnetic particles. Simultaneously one can measure the DNA end-to-end distance. Special DNA hairpin templates allowed to characterize different parameters of the DNA unwinding reaction such as the unwinding velocity, the length of unwound DNA (processivity) or the influence of forces. From this mechanistic models about the functions of the helicases could be obtained. T-Antigen is found to be one of the slowest and most processive helicases known so far. In contrast to prokaryotic helicases, the unwinding velocity of T-Antigen shows a weak dependence on the applied force. Since current physical models for the unwinding velocity fail to describe the data an alternative model is developed. The investigated RecQ helicases are found to unwind and close short stretches of DNA in a repetitive fashion. This activity is shown for the first time under external forces. The experiments revealed that the repetitive DNA unwinding is based on the ability of both enzymes to switch from one single DNA strand to the other. Although RecQ2 and RecQ3 perform repetitive DNA unwinding, both enzymes differ largely in the measured DNA unwinding properties. Most importantly, while RecQ2 is a classical helicase that unwinds DNA, RecQ3 mostly rewinds DNA duplexes. These different properties may reflect different specific tasks of the helicases during DNA repair processes. To obtain high spatial resolution in DNA unwinding experiments, the experimental methods were optimized. An improved and more stable magnetic tweezers setup with sub-nanometer resolution was built. Additionally, different methods to prepare various DNA templates for helicase experiments were developed. Furthermore, the torsional stability of magnetic particles within an external field was investigated. The results led to selection rules for DNA-microsphere constructs that allow high resolution measurementsJeder Organismus ist bestrebt, die genetischen Informationen intakt zu halten, die in seiner DNA gespeichert sind. Dies wird durch präzise gesteuerte zelluläre Prozesse wie DNA-Replikation, -Reparatur und -Rekombination verwirklicht. Ein wesentlicher Schritt ist dabei das Entwinden von DNA-Doppelsträngen zu Einzelsträngen. Diese chemische Reaktion wird von Helikasen durch die Hydrolyse von Nukleosidtriphosphaten katalysiert. Obwohl bei allen Helikasen bestimmte Aminosäuresequenzen hoch konserviert sind, können sie sich in Eigenschaften wie Struktur, Funktion oder DNA Substratspezifität stark unterscheiden. Gegenstand der vorliegenden Arbeit ist es, die Entwindungsmechanismen von drei verschieden Helikasen aus zwei unterschiedlichen Organismen zu untersuchen, die sich in ihrer Struktur sowie ihrer Funktion unterscheiden. Es handelt sich dabei um die replikative, hexamerische Helikase Large Tumor-Antigen (T-Antigen) vom Simian-Virus 40 und die DNA-Reparatur-Helikasen RecQ2 und RecQ3 der Pflanze Arabidopsis thaliana. Um DNA-Entwindung in Echtzeit zu untersuchen, wird eine biophysikalische Einzelmolekültechnik, die \"Magnetische Pinzette\", verwendet. Mit dieser Technik kann man ein DNA-Molekül, das an ein magnetisches Partikel gebunden ist, strecken und gleichzeitig dessen Gesamtlänge messen. Mit speziellen DNA-Konstrukten kann man so bestimmte Eigenschaften der Helikasen bei der DNA-Entwindung, wie z.B. Geschwindigkeit, Länge der entwundenen DNA (Prozessivität) oder den Einfluß von Kraft, ermitteln. Es wird gezeigt, dass T-Antigen eine der langsamsten und prozessivsten Helikasen ist. Im Gegensatz zu prokaryotischen Helikasen ist die Entwindungsgeschwindigkeit von T-Antigen kaum kraftabhängig. Aktuelle Modelle sagen dieses Verhalten nicht vorraus, weshalb ein alternatives Modell entwickelt wird. Die untersuchten RecQ-Helikasen zeigen ein Entwindungsverhalten bei dem permanent kurze Abschnitte von DNA entwunden und wieder zusammengeführt werden. Dieses Verhalten wird hier zum ersten Mal unter dem Einfluß externer Kräfte gemessen. Es wird gezeigt, dass die permanente Entwindung auf die Fähigkeit beider Helikasen, von einem einzelen DNA-Strang auf den anderen zu wechseln, zurückzuführen ist. Obwohl RecQ2 und RecQ3 beide das Verhalten des permanenten Entwindens aufzeigen, unterscheiden sie sich stark in anderen Eigenschaften. Der gravierendste Unterschied ist, dass RecQ2 wie eine klassische Helikase die DNA entwindet, während RecQ3 eher bestrebt ist, die DNA-Einzelstränge wieder zusammenzuführen. Die unterschiedlichen Eigenschaften könnten die verschieden Aufgaben beider Helikasen während DNA-Reparaturprozessen widerspiegeln. Weiterhin werden die experimentellen Methoden optimiert, um möglichst hohe Auflösungen der Daten zu erreichen. Dazu zählen der Aufbau einer verbesserten und stabileren \"Magnetischen Pinzette\" mit sub-nanometer Auflösung und die Entwicklung neuer Methoden, um DNA Konstrukte herzustellen. Außerdem wird die Torsions\\-steifigkeit von magnetischen Partikeln in externen magnetischen Feldern untersucht. Dabei finden sich Auswahlkriterien für DNA-gebundene magnetische Partikel, durch die eine hohe Auflösung erreicht wird
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Bailey, Scott. "Structural investigations of the Bacillus subtilis SPP1 phage G39P helicase inhibitor loading protein." Thesis, University of Sheffield, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246919.
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Langland, Gregory Todd. "Interaction Between the BLM Helicase and the DNA Mismatch Repair Protein, MLH1." University of Cincinnati / OhioLINK, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052316756.
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Brüning, Jan-Gert. "Underpinning replication of protein-bound DNA by the accessory replicative helicase Rep." Thesis, University of York, 2015. http://etheses.whiterose.ac.uk/8220/.
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Accurate DNA replication must occur prior to every cell division. However, replication forks often stall at sites of DNA damage and protein-DNA complexes. If not removed, these blocks can threaten the viability of both daughter cells by preventing the completion of genome duplication or by targeting of blocked forks by recombination enzymes that can result in gross chromosomal rearrangements and genome instability. The importance of minimising fork blockage has resulted in cells evolving repair systems to remove lesions from DNA whilst accessory replicative helicases can underpin replication fork movement through hard-to-replicate sites including protein-DNA complexes. This thesis investigates the Escherichia coli accessory replicative helicase Rep. It is shown that efficient recruitment of Rep to the replisome via an interaction with the replicative helicase DnaB is dependent on the extreme Rep C terminus. This work also indicates that the DnaB C terminus is necessary for this interaction. Secondly, this work determines the function of the 2B subdomain, a conserved feature of Superfamily 1A (SF1A) helicases. Characterisation of a Rep mutant lacking this domain (RepΔ2B) showed greatly reduced levels of protein displacement from DNA, indicating a central role of the 2B subdomain in the removal of nucleoprotein blocks. Complementation of this mutation by a 2B subdomain of the homologous helicase UvrD supports the idea that the accessory replicative helicase function of Rep is dependent on a 2B subdomain. These data also demonstrate that the function of 2B subdomains is conserved among other SF1A helicases. Previous work had also shown that the 2B subdomain of SF1A helicases is flexible. Mutations in the hinge that connect the 2B subdomain to the rest of the helicase resulted in activation of DNA helicase activity and increased levels of nucleoprotein removal from single-stranded (ss) and double-stranded (ds) DNA. These data shed new light on how translocation along DNA is coupled to protein displacement during helicase catalysis, a conserved function of many helicases. A model is proposed where ATP hydrolysis is closely linked to conformational changes of the 2B subdomain of Rep, facilitating protein displacement by Rep.
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Chou, Ta-Chung. "The molecular role of the Saccharomyces cerevisiae DNA helicase Srs2 during meiosis." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/8446/.
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Lee, Myung Soo. "Studies on the DNA helicase activities of the Escherichia coli primosome : involved in DNA replication fork movement /." Access full-text from WCMC, 1989. http://proquest.umi.com/pqdweb?did=744115351&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Bernard, Emmanuelle Alexa. "Cloning and characterisation of the Xenopus laevis bloom's protein." Thesis, University of Sussex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367351.
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Paes, Hazel Margaret. "The kinetics of DNA triple helices." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242691.
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Fletcher, Ryan James. "Structural and biochemical studies of mini-chromosomal maintenance proteins /." Connect to full text via ProQuest. IP filtered, 2005.
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Thesis (Ph.D. in Biochemistry and Molecular Genetics) -- University of Colorado, 2005.Typescript. Includes bibliographical references (leaves 84-97). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Meistermann, Isabelle. "DNA major groove recognition by supramolecular helicates." Thesis, University of Warwick, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246782.
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Yu, Jei-Hwa. "MAPKs regulate nuclear import of human papillomavirus type 11 replicative helicase E1." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/yu.pdf.
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Zecevic, Alma. "Role of WRN helicase in repair of chromate induced DNA damage : final version." View abstract/electronic edition; access limited to Brown University users, 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:3318377.
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Burrage, Joseph. "Analysis of the function of LSH in DNA damage repair." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/9416.
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DNA damage from both normal metabolic activities and environmental factors such as UV and radiation can cause as many as 1 million individual lesions to the DNA per cell per day (Lodish et al 2004). Cells respond to this continuous damage by employing many, highly efficient DNA repair mechanisms and undergo apoptosis when normal DNA repair fails. Of the many types of DNA damage that can occur, double strand breaks (DSBs) are the most toxic (Featherstone & Jackson 1999). A single unrepaired DSB is enough to induce cellular apoptosis and several mechanisms have developed to repair DSBs. The recognition, signalling and repair of DSBs involve large multi-‐subunit complexes that bind to both the DNA and modified histone tails, which require modification of the chromatin in order to access their bind sites and function effectively (Allard et al 2004). Consequently several chromatin-‐remodelling proteins have been implicated in DSB repair (van Attikum et al 2004, Chai et al 2005). LSH (Lymphoid specific helicase) is a putative chromatin-‐remodelling enzyme that interacts with DNA methyltransferases and has been connected to DNA methylation (Myant & Stancheva, 2008). Knockouts of LSH or its homologues in A. thaliana and M. musculus show a reduction in DNA methylation of 60-‐70% (Jeddeloh et al 1999, Dennis et al 2001). However in addition to this phenotype, knockout A. thaliana also have an increased sensitivity to DNA damage (Shaked et al 2006). A homologue of LSH has also been identified in S. cerevisiae, which interacts with known repair proteins (Collins et al 2007) and may be involved in DSB repair. Although the majority of Lsh-‐/-‐ mice die shortly after birth, 40% of the line produced by Sun et al survive and show unexplained premature aging (Sun et al 2004). As premature aging is a hallmark of increased acquisition of DNA damage there is the possibility of a conserved role for LSH in mammalian DNA damage repair. Here I show that LSH depleted mammalian cells have an increased sensitivity specifically to DSB inducing agents and show increased levels of apoptosis. Further analysis shows that cells lacking LSH repair DSBs slower, indicating a novel role for LSH in mammalian repair of DSB. I performed an in depth analysis of the DSB defects in LSH depleted cells in an attempt to elucidate the function of LSH in DSB repair. I found that LSH depleted cells can correctly recognise DSBs but recruit downstream signalling and repair factors, such as γH2AX, less efficiently. I show that reduced recruitment of downstream DSB repair factors is not accompanied by extended cell cycle checkpoint signalling. This suggests that LSH depleted cells continue through the mitosis with unrepaired DSBs, which most likely leads to apoptosis and the increased sensitivity to DSB inducing agents. These experiments also showed that recruitment of DSB signalling and repair factors is not impaired equally at all breaks, and I present a model system created to quantitatively compare individually breaks between WT and LSH depleted cells to identify DSB that require LSH for efficient repair. I also preformed an analysis of Lsh-/- MEFs containing WT or catalytic null mutant LSH rescue constructs and I show that WT but not catalytic null LSH can restore efficient DSB repair. These studies identify a novel role for LSH in mammalian DSB repair and demonstrate the importance of its catalytic activity.
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Hunt, Laura. "Investigating the role of the Srs2 DNA helicase during meiosis in S. cerevisiae." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/19942/.
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During cell division, duplicated chromosomes must be segregated faithfully to prevent aneuploidy in daughter cells. In meiosis, there are two rounds of division following a single round of DNA replication. In the first meiotic division, crossovers formed between homologous chromosomes, via homologous recombination, ensure correct DNA segregation. Homologous recombination is intitated by DNA double-strand breaks (DSBs), which are processed to form single-stranded DNA that can invade donor duplexes to effect repair. In yeast, formation of nucleoprotein-filaments (NPFs) by RecA homologues Rad51 and Dmc1 promotes strand invasion into sister chromatids or homologous chromosomes, respectively. I investigated the meiotic role of Srs2, a multi-functional DNA helicase that is thought to regulate mitotic strand invasion via promotion of Synthesis-Dependent Strand Annealing to prevent hyper-recombination. To investigate meiotic phenotypes of srs2 strains, including deficient meiotic progression and reduced spore viability, I analysed nuclear and spindle pole body (SPB) division of spread chromatin. I found a significant increase in single nucleus cells with divided SPBs in srs2, suggesting cells are attempting to progress into second meiosis despite the nucleus failing to divide. Using strains with integrated TetO repeats and TetR-GFP to observe division at a single chromosome level, I conclude that homologues and sister chromatids are moving apart even when the nucleus fails to divide. Immunofluorescence of Rad51, in srs2 cells, revealed bright Rad51 foci appearing as aggregates under standard microscopy, which colocalise with RPA. These are dependent on SPO11, NDT80 and Rad51 strand invasion activity, but independent of MEK1 and SAE2, suggesting the meiotic phenotype is related to DSB formation and pachytene exit but independent of DSB resection or inter-homologue strand invasion. Interestingly, a partial rescue is observed when MRX complex formation is hindered. To determine whether Rad51 aggregation occurs at known recombination hotspots, I have prepared strains for ChIPSeq to analyse any alterations in the distribution of Rad51 along the DNA. Finally, the implications for the role of Srs2 during meiosis raised by these novel observations will be discussed.
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Litman, Rachel. "Characterization of the BACH1 Helicase in the DNA Damage Response Pathway: a Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/329.
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DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability.
One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes.
The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.
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Leon, Ronald P. "Structural and functional analysis of MCM helicases in eukaryotic DNA replication /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Biophysics & Genetics, Program in Molecular Biology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 90-98). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;