Dissertations / Theses on the topic 'DNA interstrand crosslinks'

DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process.
DNA-skadande ämnen är vanligt i cancerbehandling, då snabbt växande celler, såsom cancerceller är betydligt känsligare än normala celler för DNA skador. En grupp av ämnen som vanligen används i cancerbehandling är korsbindare av DNA. Dessa ämnen kommer reagera två gånger med DNA och skapa två bindningar mitt emot varandra. DNA strängen, som består av två delar, måste kunna separeras och kopieras (replikation) på ett tillförlitligt sätt för att cellerna ska kunna dela sig och bli flera. DNA strängen måste också kunna dela sig och bli avläst rätt för att nya proteiner ska kunna bildas (transkription). När korsbindarna har bundit till DNA strängarna, hindrar detta deras separation och därigenom förhindras även avläsningen och kopieringen.  För att göra undersökningarna av DNA korsbindande ämnen ännu lite svårare, så ger korsbindare flera olika typer av skador. Dels kan det bli flera olika typer av korsbindningar, både mellan två DNA-strängar (ICL) vilket är den farligaste och mest svårreparerade typen, men det kan också ske inom samma DNA-sträng (intrastrand crosslink) eller mellan en DNA-sträng och ett protein (DNA-protein crosslink). Korsbindare kan även bilda enbindningsskador (monoaddukt), vilket innebär den bara binder en gång till DNA. För att cellen ska kunna överleva, så måste den reparera skadorna och ta bort korsbindningen eller monoaddukten. Hur detta sker i människor är inte helt klarlagt men det verkar som det sker i flera steg. Till att börja med klipps DNA sönder i ena strängen på båda sidorna om korsbindningen, detta gör att den kvarvarande delen av korsbindningen kan böjas bort. Därefter kommer cellen att skapa nytt DNA för att fylla mellanrummet som bildats. Cellen använder sig av den andra DNA strängen som mall för att sätta in rätt DNA baser, men i fallet med korsbindande ämnen så är även den strängen skadad och därför finns det en stor risk för att fel DNA baser sätts in och då uppstår mutationer. Nästa steg är att klippa den kvarvarande delen av korsbindningen, även denna gång skapas ett mellanrum som måste fyllas med nya baser. Den första artikeln i avhandlingen handlar om att försöka reda ut om det är ICLen eller monoaddukten som är orsak till olika effekter som påträffas efter behandling med korsbindande ämnen. Det vi fann var att även om det bara var från ICLs som vi kunde mäta en effekt på replikationen, så fick vi nästan lika stark effekt från monoaddukterna, som från ICL, för en av de vanligast använda markörerna (kännetecknen) för båda DNA strängarna var brutna på samma ställe (dubbelstränsbrott). Detta berodde dock inte på att även monoaddukterna skapade dubbelsträngsbrott, utan på att markören vi använde var ospecifik. Vi fann även att även om ICLs har mycket större effekt än monoaddukten på cellens överlevnad m.m., så kan man inte bortse ifrån effekten av monoaddukten och att den troligen har en betydande roll för de korsbindande ämnen som endast ger en liten del ICLs. I artikel två har vi utvecklat en ny metod, som gör det möjligt att mäta hur många ICLs som bildas vid en viss dos av de korsbindande ämnen vi undersöker. Vi kan även mäta hur fort ICLerna kan repareras i mänskliga celler med hjälp av metoden. Tack vare en kombination av våra mätningar och med hjälp av datorsimuleringar, kunde vi räkna ut hur många ICLs som bildades per dos för tre vanliga korsbindare. Vi kunde även visa att 50 % av ICLen har påbörjat reparationen och kommit så långt att de var bortklippta från ena stängen inom 3 timmar efter behandlingen. I artikel tre undersöker vi vilka proteiner som är inblandade i den tidiga delen av ICL reparationen, alltså fram till och med att celler klipper ut korsbindningen på båda sidorna om skadan i ena strängen. Här visar vi att celler som är defekta i reparationsprotein kallat XPA, har en betydligt långsammare borttagning av ICLer än vad båda normala celler och celler defekta i reparationsprotein XPE har. Vi visar även att detta inte påverkar cellens replikationshastighet, eller har någon effekt på cellens överlevnad. Den fjärde artikeln handlar om acetaldehyd, som bildas när alkohol förbränns i kroppen. Acetaldehyd har föreslagits bilda ICL och därför undersökte vi vilka effekter den har på cellerna. Vi visar i den här artikeln att det krävs nysyntes av DNA för att acetaldehyd ska leda till dubbelsträngsbrott. Celler kan reparera dessa dubbelsträngsbrott med hjälp av reparationssystem, som kallas homolog rekombination, men att reparationen ibland blir felaktig. I den femte och sista artikeln i avhandligen undersöker vi ett av de vanligast föreslagna proteinen för att sköta klippningen av DNA (ERCC1/XPF) och hur den är inblandad i reparationen av korsbindningar. Vi kan här visa att även det krosbindande ämnet mitomycin C bromsar replikationshastigheter och att ERCC1/XPF är nödvändigt för att kunna fullfölja homolog rekombination av ICLs.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Submitted.

Despite improvements in tumour response and survival following platinum based therapy in ovarian cancer, both intrinsic and acquired drug resistance remain a significant problem. Mechanisms of acquired drug resistance have been studied extensively in ovarian cancer cells in culture following repeated drug exposure however the relevance of these mechanisms to the clinical situation still needs to be clearly defined. Studies in clinical material have been hampered by the unavailability of sensitive methods to detect the critical drug-induced effects in individual cells. Recently, a modification of the single cell gel electrophoresis (comet) assay has been developed which allows for the first time the sensitive detection of DNA interstrand crosslinks in both tumour and normal cells derived directly from clinical material. In this study the role of DNA crosslink repair as a potential mechanism of clinical resistance to platinum drugs has been examined. Methods for isolating ovarian tumour cells and mesothelial (normal) cells from ascitic fluid, and to prepare a single cell suspension from primary ovarian tumour tissue from surgery have been established. Cells treated ex vivo with cisplatin were examined for crosslink formation and repair (unhooking) using the comet assay. No significant difference in the peak level of crosslinking was observed between tumour and mesothelial cells from an individual patient, or between patients either untreated or previously treated with platinum-based therapy. In the majority of tumours from nine untreated patients little or no repair of crosslinks was evident at 24 hours (mean of 13.6% repair) with seven patients showing less than 10% repair. In contrast, in the majority of ten treated patients a high level of repair was observed in tumour cells (mean 46.5%), with greater than 70% repair in four at 24 hours. Increased interstrand crosslink repair was also observed in a cisplatin acquired resistant human ovarian tumour cell line. Differences in gene expression pattern between the sensitive and resistant cell lines were compared using microarray analysis. The expression of a number of genes, including ERCC1, were consistently elevated in the resistant cell line, which was confirmed using RT-PCR.

Platinum-based chemotherapeutics are used for the treatment of a wide range of cancers and a number of attempts have been made toward developing compounds with better cellular stability and similar or enhanced cytotoxicity as compared to their predecessors. The first part of the work reported here focuses on the cellular effects of the metabolically stable dinuclear platinum compound, BBR3610-DACH. Comet assay showed this compound to form interstrand crosslinks, a highly toxic DNA lesion in HCT116 cells, at equimolar concentrations to its parental compound, BBR3610. Cell cycle studies showed that BBR3610-DACH causes G1/S and G2/M cell cycle arrest with S phase depletion, which was p21 dependent and partially p53 dependent in contrast to BBR3610 which showed initial S phase accumulation followed by a classical G2/M arrest. BBR3610-DACH-induced G1/S and G2/M cell cycle arrest interestingly was found to be independent of the DNA damage response mediated via the activation of ATM and ATR kinases. Also, the cell cycle arrest culminated in apoptosis, although apparently through a non-canonical pathway. The second project explores the cellular effects of trans-4-NBD which is a fluorescent derivative of transplatin. Like cisplatin, trans-4-NBD induced interstrand crosslinks in HCT116 cells as detected by the comet assay. Treatment with trans-4-NBD showed a G2/M arrest in HCT116 cells and a transient S phase accumulation in A2780 cells, with a marked increase in p53 and p21 protein levels. A robust apoptotic response is also seen via caspase activation and PARP cleavage in both the cell lines. Finally, the focus is shifted toward the nucleolar targeting platinum complex, TriplatinNC. Confocal studies in TriplatinNC-treated HCT116 and A2780 cells showed disruption of rRNA transcription as an early event followed by a robust G1 cell cycle arrest. Apoptotic induction was observed with the onset of cellular morphological changes and apparent caspase activation which was independent of the p53 status of the cells. Overall, these studies explore novel platinum based compounds that show promising anti-cancer activities by affecting various facets of cellular signaling.

Genomes are constantly challenged by agents that promote DNA damage, with interstrand crosslinks (ICLs) representing a particularly dangerous lesion. Ongoing work in the Wilkinson laboratory aimed at identifying novel agents that target Trypanosoma brucei, the causative agent of African trypanosomiasis, identified several prodrugs that once activated form ICLs in this protozoan parasite. To understand the complexity of ICL repair systems that T. brucei employs to resolve such damage, a variety of null mutant lines were generated that lack activities postulated to fix such lesions. Phenotypic screens using various DNA damaging agents revealed that TbMRE11, TbEXO1, TbCSB, TbCHL1, TbFAN1, TbBRCA2 and TbRAD51 all help to resolve ICLs, implicating components of the homologous recombination, nucleotide excision repair and mismatch repair pathways in resolving this form of damage: This approach demonstrated that components of the translesion synthesis pathway (TbREV2 and TbREV3) do not play a significant role in ICL repair. In many organisms, nucleases belonging to the SNM1/PSO2 family play a key and specific role in the repair of ICLs with this property extending to the T. brucei homologue, TbSNM1. To assess whether there is a functional linkage between the DNA repair factors noted above and TbSNM1, a series of double null mutants were constructed and the susceptibility of these lines to ICL inducing agents determined. Identification of their epistatic/non-epistatic interactions revealed that T. brucei expresses at least two ICL repair systems with one pathway involving the concerted activities of TbSNM1/TbCSB/TbEXO1, that we postulate functions to repair ICLs encountered by the transcriptional machinery, while the other is centred upon TbMRE11/TbFAN1/TbEXO1 that may help resolve lesions which cause stalling of DNA replication forks. By unravelling how T. brucei repairs ICLs, specific inhibitors against key components of these pathways could be developed and used in combination with DNA damaging agents to target trypanosomal infections.

DNA interstrand crosslinks (ICLs) can be induced by multiple crosslinking agents and are a severe form of DNA damage that prevents strand separation during DNA replication and transcription. Failure to repair endogenous ICLs in S phase is thought to underlie the bone marrow failure syndrome Fanconi anemia, and up-regulation of ICL repair is one of the causes of tumor chemoresistance against widely used cancer drugs. In metazoans, a major pathway of ICL repair is coupled to DNA replication and requires the Fanconi anemia pathway. In most current models, collision of a single DNA replication fork with an ICL is sufficient to initiate repair. In contrast, we show that in Xenopus egg extracts, two DNA replication forks must converge on an ICL to trigger repair. When only one fork reaches the ICL, the replicative DNA helicase, CMG, fails to unload from the stalled fork, and repair is blocked. Arrival of a second fork, even when substantially delayed, rescues repair. We conclude that ICL repair requires a replication-induced X-shaped DNA structure surrounding the lesion, and we speculate how this requirement helps maintain genomic stability in S phase. Next, we focus on how psoralen-induced ICLs are repaired. Although psoralen-ICL repair still requires replication fork convergence, downstream repair events are completely different. Unlike cisplatin-ICLs, which are resolved via incisions in the parental strands with formation of double-strand breaks (DSBs), psoralen-ICLs are resolved via cleavage of a N-glycosyl bond that forms part of the ICL. Unlike cisplatin-ICL repair, psoralen-ICL repair does not require CMG helicase unloading or FANCI-FANCD2. When N-glycosyl bond cleavage is inhibited, the psoralen-ICL is repaired via FANCI-FANCD2-dependent incisions. Our work identifies a novel S phase ICL repair mechanism that is independent of the Fanconi anemia pathway and avoids DSBs.
Medical Sciences

Budding yeast Slx4 binds to the structure-specific DNA repair nucleases Slx1 and Rad1XPF-Rad10ERCC1, and it was reported that Slx4 is essential for DNA flap cleavage by Rad1XPF-Rad10ERCC1 during certain types of DNA repair in yeast. At the outset of this thesis, bioinformatic analyses identified the uncharacterised protein BTBD12 in higher eukaryotes as a putative orthologue of yeast Slx4. In the first results chapter of this thesis, I describe the identification of BTBD12-interacting proteins, including XPF-ERCC1 and SLX1. These findings led me to refer to BTBD12 as human SLX4. I found that SLX4 binds to another structure-specific nuclease MUS81-EME1, and other proteins involved in telomere maintenance and cell cycle progression. The remainder of this chapter describes detailed biochemical analysis of the nuclease activities associated with the SLX4 complex isolated from human cells. Work from this lab and others revealed that depletion of SLX4 from human cells using siRNAs causes defects in the repair of DNA interstrand crosslinks (ICLs). Inherited mutations in humans that reduce the efficiency of ICL repair cause Fanconi anaemia (FA). The cellular sensitivity of SLX4 depleted cells to ICLs prompted me to investigate SLX4 as a candidate FA gene. Dr. Johan de Winter (VU University Medical Center, Amsterdam) and Dr. Detlev Schindler (University of Wurzburg) had identified several patients with unclassified FA that was not caused by mutations in the FA genes known at the time. In the second results I describe characterisation of SLX4, and the SLX4 holo-complex, in cells from some of these FA patients who had bi-allelic SLX4 mutations. In three of the patients SLX4 was expressed at normal levels but was missing part of the first, and all of the second, UBZ-type putative ubiquitin-binding domain. This prompted me to investigate the function of the SLX4 UBZ domains. I found that the first, but not the second, UBZ domain of SLX4 binds to ubiquitin in vitro and targets SLX4 to sites of DNA damage in vivo. Furthermore, the first but not the second SLX4 UBZ domain appears to be required for ICL repair, demonstrating the important of correctly localising SLX4 for DNA repair. In the final chapter, I present preliminary data which suggests that SLX4 is regulated in an unusual manner in during S-phase of the cell cycle, and that SLX4 interacts with the PLK1 kinase in a phosphorylation-dependent manner.

Kazunori Yoshikiyo, Katja Kratz, Kouji Hirota, Kana Nishihara, Minoru Takata, Hitoshi Kurumizaka, Satoshi Horimoto, Shunichi Takeda, and Josef Jiricny "KIAA1018/FAN1 nuclease protects cells against genomic instability induced by interstrand cross-linking agents" PNAS 2010 107 (50) 21553-21557; published ahead of print November 29, 2010, doi:10.1073/pnas.1011081107
Kyoto University (京都大学)
0048
新制・論文博士
博士(医学)
乙第12772号
論医博第2063号
新制||医||1000(附属図書館)
30755
(主査)教授 小松 賢志, 教授 小川 誠司, 教授 松本 智裕
学位規則第4条第2項該当

DNA interstrand crosslinks (ICLs) are a potent type of DNA damage that arise as a consequence of normal cell metabolism. By covalently linking opposing strands of the double helix, ICLs block essential DNA transactions such as replication, transcription, and recombination. If unrepaired, or incorrectly repaired, ICLs can lead to gross genome instability and cell death. This cytotoxicity has been exploited in the clinic, where ICL inducing drugs are among the oldest and most widely prescribed anti-cancer therapies. However, acquired resistance is a significant limitation of these drugs, and the mechanism by which this occurs remains largely elusive. In order to develop more effective ICL-based therapies, it is imperative to first fully elucidate how healthy cells respond to and repair ICLs. Moreover, better understanding ICL repair mechanisms is necessary to fully unravel the complex DNA repair networks that govern genomic integrity, and understand the physiology of diseases such as Fanconi Anemia, which result from the inability to efficiently repair ICL lesions. Multiple mechanisms of ICL repair exist, and repair pathway choice is primarily determined by the phase of the cell cycle. In proliferating cells, the ICL repair occurs during S-phase, and in a process termed “replication coupled repair” (RCR). In contrast, slowly or non-dividing cells rely on an alternative modality of repair called “replication independent repair” (RIR). RIR is critical for homeostasis and survival in quiescent healthy cells that (for example, neurons) and in cycling cells deficient for replication coupled repair proteins (i.e. Fanconi Anemia cells). Despite its importance, little is known about RIR. This is due, in part, to the fact that ICL repair has been primarily studied in systems, such as cultured cells, that favor RCR and are therefore bias against RIR. More recently, non-replicating Xenopus cell-free extracts has emerged as a powerful system to study RIR. This system faithfully recapitulates RIR and has been instrumental in identifying DNA polymerase kappa (Pol κ) and the eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), as two critical RIR factors. However, other important RIR factors are yet to be identified. ICL repair is unique among DNA repair pathways as it harnesses proteins from diverse DNA repair pathways including, Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), and Double Strand Break Repair (DSBR). Chapter 1 provides an overview of these pathways including the types of DNA damage that each pathway responds to, key steps of the repair process, and the corresponding proteins that are involved. This chapter provides context for the rest of the thesis in which I explore the contribution of multiple DNA repair proteins on the repair of ICL lesions. In Chapter 2, I detail our studies assessing the contribution of the MMR machinery to RIR. We show that the mismatch repair sensor, MutS complex (MSH2-MSH6), is critical for ICL recognition, and the stepwise recruitment of other MMR proteins including MutL (MLH1-PMS2) and EXO1. In this chapter, I also investigate how ICL structure influences repair. I find that more distorting ICLs use an MMR-dependent ICL repair mechanism, while less distorting ICLs are repaired MMR-independently (see also Appendix A), or not repaired at all. Appendix B further explores the contribution of the MMR pathway on ICL repair in mammalian cells. Finally, in Appendix C and D we provide further evidence that RIR is fundamentally distinct from replication coupled ICL repair, as depletion of key RCR proteins from our extracts yields no phenotype. I summarize all of these findings in Chapter 3, and discuss their implications to the DNA repair field as well as the clinic, where crosslinker drugs remain a mainstay in the treatment of cancer.

archives@tulane.edu
BRCA1 faithfully repairs damaged DNA by promoting homology-directed repair (HDR). Loss of Brca1 and other HDR genes are incompatible with embryonic viability and cause severe genomic instability. Cells lacking BRCA1 are sensitive to cellular stresses such as DNA damage caused by ionizing radiation (IR). Homozygous loss of Brca1 is embryonic lethal in mice, and the few tissue-specific knockouts generated develop abnormally. Therefore, we created an inducible Cre mouse model to study Brca1 loss in all adult mouse tissues allowing for examination of viability, longevity, and stress response in the absence of HDR and the importance of HDR in different tissues of an adult mouse. After validating the inducible Cre system using a reporter allele in mice, we generated mice with alleles of the inducible Cre system and floxed Brca1 alleles. Cre was induced in adult mice at ten weeks of age, resulting in extensive, widespread deletion of Brca1. Contrary to the embryonic lethality observed in all previously tested germline Brca1 knockout mouse models, adult mice with Brca1 deletion displayed no overt phenotypes. Brca1Δ/Δ mice showed extensive, widespread deletion of Brca1 and survived up to 1 year after Brca1 recombination. Targeted, high-depth sequencing of recombined tissues indicated mutations accumulated in both the mammary gland and the intestine. However, only the mammary gland had an HDR deficiency signature. Next, we examined Brca1Δ/Δ mice survival after exposure to ionizing radiation and mitomycin C (MMC). Surprisingly, Brca1Δ/Δ mice responses are DNA damage specific. Brca1Δ/Δ mice deficient for HDR showed no increased sensitivity to IR but died four to eight days following MMC exposure. Our results show that BRCA1 is not required for long-term viability or DNA double-strand break repair, but BRCA1 is essential for DNA crosslink repair to maintain viability in an adult mouse.
1
JoyOlayiwola

The repair of DNA interstrand crosslinks (ICLs) involves the concerted action of multiple DNA repair pathways. In eukaryotes, ICLs are sensed and repaired by replication-coupled or replication-independent mechanisms in which PSO2/SNM1 is essential for unhooking an ICL. Previous work has shown that ICL-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA (mtDNA), and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mtDNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. In the first part of this study, we provide direct evidence that the S. cerevisiae nuclear-encoded Pso2 is a dual-localized ICL repair factor: it is imported into mitochondria and the nucleus, and its abundance increases in the mitochondria following treatment with DNA damaging agents. We reveal that Pso2 contains one mitochondrial targeting sequence (MTS) and two nuclear localization signals (NLS1 and NLS2) in its N-terminus, although NLS1 resides within the MTS. While MTS is necessary to guide the passage of Pso2 into the mitochondrial matrix, either NLS1 or NLS2 is sufficient for its import into the nucleus, implying that the two NLS motifs are functionally redundant. Ablation of MTS abrogated mitochondrial import of Pso2, and concomitant enrichment in the nucleus. Strikingly, mutations that render both NLS1 and NLS2 inactive, blocked nuclear import of Pso2; at the same time, increased its mitochondrial localization, consistent with the notion that the relationship between MTS and NLS motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 to two genome-bearing organelles is discussed In the second part of this study, we combined a range of methods and approaches to determine the cell-intrinsic and extrinsic factors that regulate the intracellular distribution and function of Pso2 in ICL repair. Indeed, these approaches demonstrated that Pso2 is modified by ubiquitination and phosphorylation (but not SUMOylation) under normal growth conditions. Notably, we found that SUMOylation of Pso2 and its subsequent import into the mitochondria was induced by methyl methanesulfonate (MMS), but not cisplatin. We also demonstrate that the levels of MMS-induced Pso2 SUMOylation remain relatively constant throughout the synchronized cell cycle. Mutation of lysine residues (K97 and K575) within the SUMO consensus motifs in Pso2 abolished its import into the mitochondria. We further show that SIZ1 and SIZ2 SUMO E3 ligases act redundantly to catalyze SUMOylation of Pso2, while the former plays a dominant role in this process. Quite unexpectedly, we found that PSO1 (but not PSO2) was essential for attenuation of MMS-induced cell death; this implies that they function in non-overlapping ICL repair pathways or the former is epistatic to the latter. The findings reveal novel mechanistic insights into the regulation and intracellular distribution of Pso2 and shed new light on its function in DNA repair.

The Fanconi anemia (FA) pathway is a replication-dependent DNA repair mechanism which is essential for the removal of interstrand crosslink (ICL) DNA damages in higher eukaryotes (Moldovan and D’Andrea, 2009). Malfunctions in this highly regulated repair network lead to genome instability (Deans and West, 2011). Pathological phenotypes of the disease FA which is caused by mutations in the eponymous pathway are very heterogeneous, involving congenital abnormalities, bone-marrow failure, cancer predisposition and infertility (Auerbach, 2009). The FA pathway comprises a complex interaction network and to date 16 FA complementation groups and associated factors have been identified (Kottemann and Smogorzewska, 2013). Additionally, components of nucleotide excision repair (NER), homologous recombination repair (HRR), and translesion synthesis (TLS) are involved and coordinated by the FA proteins (Niedzwiedz et al., 2004; Knipscheer et al., 2009). One of the FA proteins is the DEAH helicase FANCM. In complex with its binding partners FAAP24 and MHF1/2 it binds the stalled replication fork and activates the FA damage response (Wang et al., 2013). However, the exact steps towards removal of the ICL damage still remain elusive. To decipher the underlying process of FA initiation by FANCM, this thesis mainly focuses on the archaeal FANCM homolog helicase-associated endonuclease for fork-structured DNA (Hef). Hef from the archaeal organism Thermoplasma acidophilum (taHef) differs from other archaeal Hef proteins and exclusively comprises an N-terminal helicase entity with two RecA and a thumb-like domain while others additionally contain a nuclease portion at the C-terminus. I solved the crystal structure of full-length taHef at a resolution of 2.43 Å. In contrast to the crystal structure of the helicase domain of Hef from Pyrococcus furiosus (pfHef), taHef exhibits an extremely open conformation (Nishino et al., 2005b) which implies that a domain movement of the RecA-like helicase motor domains of 61° is possible thus highlighting the flexibility of helicases which is required to translocate along the DNA. However, small-angle x-ray scattering (SAXS) measurements confirm an intermediate conformation of taHef in solution indicating that both crystal structures represent rather edge states. Most importantly, proliferating cell nuclear antigen (PCNA) was identified as an interaction partner of Hef. This interaction is mediated by a highly conserved canonical PCNA interacting peptide (PIP) motif. Intriguingly, the presence of PCNA does not alter the ATPase nor the helicase activity of taHef, thus suggesting that the interaction is entirely dedicated to recruit taHef to the replication fork to fulfill its function. Due to a high level of flexibility the taHef-taPCNA complex could not be crystallized and therefore SAXS was utilized to determine a low-resolution model of this quaternary structure. This newly discovered PCNA interaction could also be validated for the eukaryotic FANCM homolog Mph1 from the thermophilic fungus Chaetomium thermophilum (ctMph1). As the first step towards the characterization of this interaction I solved the crystal structure of PCNA from Chaetomium thermophilum (ctPCNA). Furthermore, it was possible to achieve preliminary results on the putative interaction between the human proteins FANCM and PCNA (hsFANCM, hsPCNA). In collaboration with Detlev Schindler (Human Genetics, Würzburg) and Weidong Wang (National Institute on Aging, Baltimore, USA) co-immunoprecipitation (CoIP) experiments were performed using hsFANCM and hsPCNA expressed in HEK293 cells. Although an interaction was reproducibly observed in hydroxyurea stimulated cells further experiments and optimization procedures are required and ongoing
Der Fanconi Anämie (FA) Signalweg ist ein replikationsabhängiger DNA-Reparaturmechanismus, der grundlegend zur Beseitigung von DNA-Schäden in Form von intermolekularen Quervernetzungen (ICL) beiträgt (Moldovan and D’Andrea, 2009). Fehlfunktionen in diesem stringent regulierten Reparaturnetzwerk führen somit zu Genominstabilität (Deans and West, 2011). Der pathologische Phänotyp der Krankheit FA, die durch Mutationen in dem gleichnamigen DNA-Reparatur Signalweg verursacht wird, ist sehr heterogen und umfasst angeborene Deformationen, Knochenmarksversagen, eine erhöhte Tumor Disposition sowie Infertilität (Auerbach, 2009). Der FA Mechanismus ist ein komplexes Netzwerk und bisher wurden 16 FA Komplementationsgruppen sowie weitere beteiligte Faktoren identifiziert (Kottemann and Smogorzewska, 2013). Zusätzlich sind Komponenten der Nukleotid-Exzisionsreparatur (NER), der homologen Rekombinationsreparatur (HRR) und Transläsionssynthese (TLS) involviert, die durch FA Proteine koordiniert werden (Niedzwiedz et al., 2004; Knipscheer et al., 2009). Eines der FA Proteine ist die DEAH Helikase FANCM. Im Komplex mit seinen Interaktionspartnern FAAP24 und MHF1/2 bindet FANCM an die durch den ICL Schaden zum Stillstand gekommene Replikationsgabel und aktiviert die FA Schadensantwort (Wang et al., 2013). Die weiteren Schritte, die zur Entfernung des ICL Schadens führen, sind jedoch weitestgehend ungeklärt. Zur Aufklärung der Initiation des FA Mechanismus und der Rolle, die das FANCM dabei spielt, wurde in dieser Arbeit hauptsächlich das archaische FANCM Homolog Helicase-associated Endonuclease for Fork-structured DNA (Hef) analysiert. Hef aus dem archaischen Organismus Thermoplasma acidophilum (taHef) unterscheidet sich von anderen archaischen Hef Proteinen und besteht ausschließlich aus einem N-terminalen Helikase-Abschnitt mit zwei RecA und einer thumb-like Domäne, während andere Hef Proteine am C-Terminus zusätzlich eine Nuklease-Domäne besitzen. Ich habe die Kristallstruktur des taHef Proteins bei einer Auflösung von 2,43 Å gelöst. Im Gegensatz zur Kristallstruktur eines vergleichbaren Hef-Konstruktes aus Pyrococcus furiosus (pfHef) (Nishino et al., 2005b) liegt in taHef eine extrem offene Konformation der beiden RecA-Domänen vor, was impliziert, dass eine Bewegung der RecA-ähnlichen Helikase Motordomänen um 61° möglich ist und zudem die zur Translokation entlang der DNA notwendige Flexibilität von Helikasen verdeutlicht. Messungen mittels Kleinwinkelröntgenstreuung (SAXS) deuten hingegen auf eine intermediäre Konformation des taHef Proteins in Lösung hin, wodurch beide Kristallstrukturen als eher Randzustände angesehen werden können. Besonders hervorzuheben ist, dass das Protein Proliferating Cell Nuclear Antigen (PCNA) als Hef Interaktionspartner identifiziert wurde. Diese Interaktion wird durch ein hoch-konserviertes kanonisches PCNA Interaktionspeptid-Motiv vermittelt. Interessanterweise beeinflusst PCNA aber weder die ATPase noch die Helikase Aktivität von taHef, was darauf hindeutet, dass diese Interaktion nur zur Rekrutierung des Hef Proteins zur Replikationsgabel dient. Wegen des hohen Maßes an Flexibilität konnte der taHef-taPCNA Komplex nicht kristallisiert werden, wohingegen SAXS Messungen erfolgreich waren und ein Model bei niedriger Auflösung konnte erhalten werden. Diese nachgewiesene Interaktion zwischen Hef und PCNA konnte auch für das eukaryotische FANCM Homolog Mph1 aus dem thermophilen Pilz Chaetomium thermophilum (ctMph1) bestätigt werden. Als ersten Schritt zur Charakterisierung dieser Interaktion habe ich die Kristallstruktur von PCNA aus Chaetomium thermophilum (ctPCNA) gelöst. Weiterhin war es möglich, vorläufige Resultate bezüglich der mutmaßlichen Interaktion zwischen den humanen Proteinen FANCM und PCNA (hsFANCM, hsPCNA) zu erhalten. In Kooperation mit Detlev Schindler (Humangenetik, Würzburg) und Weidong Wang (National Institute on Aging, Baltimore, USA) wurden Co-Immunopräzipitations-Experimente (CoIP) mit humanem FANCM und humanem PCNA aus HEK293-Zellen durchgeführt. Obwohl eine Interaktion in Hydroxyurea-stimulierten Zellen reproduzierbar nachgewiesen werden konnte, sind weitere Experimente notwendig, um diese Interaktion zu charakterisieren

碩士
高雄醫學院
天然藥物研究所
85
The pyrrolo[2,1-c][1,4]benzodiazepine ( PBDs ) are diverse and unusual group of antitumor antibiotics produced by streptomyces species. These biosynthetically derived compounds are well known for inhibiting DNA replication on account of DNA-antibiotics adducts through their C-11 carbinolamine functionality. However, theirclinical application has been limited by various dose- limiting toxicity. For example,anthramycin produce cardiotoxicity. Pervious studies have shown that a head-to-head synthetic antitumor agent incorporating a pair of DNA alkylating units based on the monofunctional PBDs is more potent and less cardiotoxicity. To enchancing DNA sequence selectivity,we design and synthesis C2 linked ( tail-to-tail ) PBD cross-linking agents. The tail-to-tail N-methoxymethyl pyrrolo[2,1-c][1,4 ]benzodiazepine-5,11-dione dimer has been prepared, then the partial hydride reduction was used to form an imine contained tail-to-tail PBDs crosslinker.

O6-alkylguanine-DNA-alkyltransferase (AGT) has been implicated in reducing the therapeutic efficacy of alkylating agents that cause mutations and apoptosis. Certain chemotherapeutic treatments involve the use of alkylating agents; bis-chloroethylnitrosourea (BCNU) is an example of an agent that has been investigated which has been shown to introduce DNA interstrand crosslinks (ICL). AGT directly repairs O6-alkylated guanines by flipping the damaged base into the active site and irreversibly transferring the alkyl group to an active site cysteine. Previous studies revealed that human AGT could repair DNA containing an O6-2’-deoxyguanosine-heptylene-O6-2’-deoxyguanosine (O6-dG-heptylene-O6-dG) ICL in a 5’-GNC-3’ motif mimicking a hepsulfam lesion. It is not yet understood, however, how the enzyme could access the bulky ICL lesion. We synthesized and studied DNA duplexes containing O6-dG-heptylene-O6-dG ICL lesion in a 5’-GNC-3’ motif (where N is any base) and a fluorescent base incorporated at various positions to observe the extent to which DNA is denatured upon AGT binding. 6-Methyl-3-(β-D-2-deoxyribofuranosyl)pyrrolo[2,3-d] pyrimidin-2-one (pyrrolo-dC), is a base analog that fluoresces when not base-paired and exhibits little effect on DNA stability, structure and AGT repair. All DNA substrates were synthesized and AGT homologs purified and characterized using various biophysical techniques including CD, UV spectroscopy, gel electrophoresis and MS-Q-TOF. Radioactivity binding and repair assays were used to characterize ICL-AGT association. The DNA substrates exhibited B-form structure regardless of pyrrolo-dC or ICL incorporation. Furthermore, C145S and R128A variants of AGT and wild type Ada-C (from E. coli) display no repair of the ICL DNA. The arginine “finger” (R128) appears to play an essential role in repair but not in damage detection. Incorporation of pyrrolo-dC did not specifically demonstrate the extent of DNA denaturation due to the observation of increased fluorescence when positioned near the end of the DNA double helix relative to when placed in the center of the duplex. Fluorescence studies provide a base with which to develop new AGT variants with more complex ICL DNA substrates to study their interaction and possibly provide a new method for determining binding dissociation constants without hazardous radioactive material. Better understanding of the interaction of AGT and its homologs with ICL DNA provides greater knowledge about AGT function, an enzyme that plays a role in both restoring genomic integrity and therapeutic resistance.