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. / <p>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.</p>

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 / 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 / Doctor of Medical Science / Kyoto University / DFAM

<p>DNA interstrand crosslinks provide a challenge for repair machinery given that both strands contain the lesion. Cells have evolved a sophisticated mechanism to overcome this, by recruiting proteins from several repair pathways. One protein thought to function solely in interstrand-crosslinking repair is Pso2. Pso2 deficient cells display sensitivity towards ICL agents and accumulate DNA double strand breaks upon exposure. However, Pso2 is not required for repair of DNA double strand breaks generated by other means, suggesting that these particular breaks are unique requiring Pso2 processing for successful repair. To identify what characteristics these breaks possess and what role Pso2 plays in processing theses breaks, a thorough <em>in vivo</em> and <em>in vitro </em>characterization of Pso2 was conducted.</p> <p>Pso2 was found to be a 5’-exonuclease independent of DNA structure and length but completely dependent on a 5’-phosphate. Pso2 also displayed structure-specific DNA hairpin-opening activity at the 3’ end two nucleotides from the apex. This activity was required for repair of genomic DNA capped by hairpin structures in the absence of ICL inducing agents as well those generated in response to ICL damage. The constitutively active DNA hairpin endonuclease β-CASP domain of Artemis was able to partially restore the DNA hairpin-opening deficiency and suppress the ICL defect in a <em>pso2 </em>null strain. This suggests that Pso2 acts as an endonuclease in ICL repair and that DNA hairpins may be an encountered intermediate, leading to further understanding of how this unique protein function in ICL repair as well as the repair mechanism itself.</p> / Doctor of Science (PhD)

<p>DNA interstrand cross-links (ICLs) occur when various chemical agents bind to chromosomal DNA and form a covalent bond between adjacent strands, preventing unwinding of the DNA double helix. The formation of an ICL is therefore extremely toxic to cells and necessitates quick removal and subsequent repair. Human SNM1A is a 5’-phosphate-dependent exonuclease that has been shown to be selectively involved in ICL repair; however the mechanism by which it processes ICL substrates remains unclear. To address this, our research is focused on the structural and functional characterization of SNM1A to determine this mechanism of substrate processing. In this thesis, we report the purification of human SNM1A_698-1040 as a His₆-NusA tagged protein from 4 L of <em>E. Coli</em> cell culture. This protein was found to possess 5’-phosphate-dependent exonuclease activity, and demonstrated a preference for ssDNA. Additionally, electrophoretic mobility shift assays performed with a D736A/H737A mutant suggest that the binding of SNM1A to DNA is independent of the presence of a 5’ phosphate. Collectively, these results provide insight into the mechanism of SNM1A substrate processing in ICL repair, and establish a platform for future studies of this protein.</p> / Master of Science (MSc)

<p>Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase &#950; through the Rev7 subunit and with polymerases &#954;, &#953; and &#951; in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. </p><p>In chapter 1, the solution structures of the mouse Rev1 CTD and its complex with the Pol &#954; RIR are reported, revealing an atypical four-helix bundle. Yeast two-hybrid assays were used to identify a Rev7-binding surface centered at the &#945;2-&#945;3 loop and N-terminal half of &#945;3 of the Rev1 CTD. Binding of the mouse Pol &#954; RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn &#945;-helix, with the helix stabilized by an N-terminal cap. RIR-binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a &#946;-hairpin that projects over the shallow &#945;1-&#945;2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. The combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes.</p><p>The multifaceted abilities of the Rev1 CTD are further explicated in chapter 2 where the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol &#950; complex, and the Pol &#954; RIR is reported. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a mega translesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.</p><p>Chapter 3 explores the ubiquitin-binding capacity of the FAAP20 UBZ in an effort to begin understanding its requirement for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery through recognition of monoubiquitinated Rev1. FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. Although the UBZ-ubiquitin interaction is thought to be exclusively encapsulated within the &#946;&#946;&#945; module of UBZ, it is revealed that the FAAP20-ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin-binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended &#946;-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20-ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.</p><p>Having structurally elucidated the molecular basis of the essential scaffolding function of the Rev1 CTD, the search for small molecule inhibitors of the Rev1-Rev7 interaction has been initiated toward the goal of developing novel adjuvants to DNA targeting chemotherapeutics. Screening efforts have led to the discovery of a lead compound, JH-RE-06*NaOH, that specifically targets the Rev7-binding hydrophobic pocket of the Rev1 CTD with low micromolar affinity, effectively inhibiting the Rev1-Rev7 interaction in an in vitro ELISA assay developed for high-throughput screening of small molecule libraries. With the potential for positive outcomes in future in vivo assays, we hope to develop JH-RE-06*NaOH into the first potent inhibitor of translesion synthesis in cancer patients being treated with DNA-targetng chemotherapeutics to aid in sensitization and prevention of chemoresistance development in malignancies.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19958号 / 理博第4225号 / 新制||理||1607(附属図書館) / 33054 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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