京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18176号 / 医博第3896号 / 新制||医||1003(附属図書館) / 31034 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小松 賢志, 教授 髙田 穣, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

One major pathway to overcome DNA damage induced replication blocks is translesion DNA synthesis, which is the replicative bypass of DNA damage by non-classical polymerases. For the cell to utilize translesion synthesis the non-classical DNA polymerase is recruited to sites of DNA damage, and a polymerase switch occurs between the stalled classical polymerase and the incoming non-classical polymerase. This process requires the replication accessory factor proliferating cell nuclear antigen (PCNA) and its monoubiquitination at Lys-164. To better understand the role of PCNA during translesion synthesis, I biochemically and structural characterized two PCNA mutant proteins, G178S and E113G PCNA, which are defective in translesion synthesis. The X-ray crystal structure of both mutant proteins showed a shift in an extended loop, called loop J, compared to the wild type PCNA structure. Steady state kinetic studies determined that in contrast to wild type PCNA which stimulates the non-classical polymerases, the two PCNA mutant proteins fail to stimulate the activity of the non-classical polymerase pol η. These results indicate that loop J in PCNA plays an essential role in facilitating translesion synthesis. During the structural studies of the E113G PCNA mutant protein I observed a unique PCNA structure that failed to form the characteristic PCNA ring shape structure, through traditional intersubunit interactions of domain A and domain B on neighboring subunits. Instead this non-trimeric PCNA structure formed A-A and B-B intersubunit interactions. The B-B interface is structurally similar to the A-B interface observed for the trimeric ring shaped form. In contrast the A-A interface is stabilized by hydrophobic interactions. The location of the E113G substitution is directly within this hydrophobic surface and would not be favorable in the wild type protein. This suggests that the side chain of Glu-113 promotes trimer formation by destabilizing these possible alternate subunit interactions. To biochemically and structurally characterize the impact of monoubiquitinating PCNA (Ub-PCNA), I developed an Ub-PCNA analog by splitting the protein into two self-assembling polypeptides. This analog supports cell growth and translesion synthesis in vivo, and steady state kinetic studies showed that the Ub-PCNA analog stimulates the catalytic activity of pol η in vitro. The X-ray crystal structure of Ub-PCNA showed that the ubiquitin moieties are located on the back face of PCNA. Surprisingly, the attachment of ubiquitin does not change PCNA's conformation. This implies that PCNA ubiquitination does not cause an allosteric change to PCNA, and instead facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for the non-classical polymerases.

All living organisms are made of cells, within which genetic information is stored on long strands of deoxyribonucleic acid (DNA). The DNA encodes thousands of different genes and provides the blueprint for all of the structures and activities occurring within the cell. The building blocks of DNA are the four deoxyribonucleotides, dATP, dGTP, dTTP, and dCTP, which are collectively referred to as dNTPs. The key enzyme in the production of dNTPs is ribonucleotide reductase (RNR). In the budding yeast Saccharomyces cerevisiae, the concentrations of the individual dNTPs are not equal and it is primarily RNR that maintains this balance. Maintenance of the dNTP pool balance is critical for accurate DNA replication and DNA repair since elevated and/or imbalanced dNTP concentrations increase the mutation rate and can ultimately lead to genomic instability and cancer. In response to DNA damage, the overall dNTP concentration in S. cerevisiae increases. Cell survival rates increase as a result of the elevated concentration of dNTPs, but the cells also suffer from a concomitant increase in mutation rates. When the replication machinery encounters DNA damage that it cannot bypass, the replication fork stalls and recruits specialized translesion synthesis (TLS) polymerases that bypass the damage so that replication can continue. We hypothesized that elevated dNTP levels in response to DNA damage may allow the TLS polymerases to more efficiently bypass DNA damage. To explore this possibility, we deleted all known TLS polymerases in a yeast strain in which we could artificially increase the dNTP concentrations. Surprisingly, even though all TLS polymerases had been deleted, elevated dNTP concentrations led to increased cell survival after DNA damage. These results suggest that replicative DNA polymerases may be involved in the bypass of certain DNA lesions under conditions of elevated dNTPs. We confirmed this hypothesis in vitro by demonstrating that high dNTP concentrations result in an increased efficiency in the bypass of certain DNA lesions by DNA polymerase epsilon, a replicative DNA polymerase not normally associated with TLS activity. We asked ourselves if it would be possible to create yeast strains with imbalanced dNTP concentrations in vivo, and, if so, would these imbalances be recognized by the checkpoint control mechanisms in the cell. To address these questions, we focused on the highly conserved loop2 of the allosteric specificity site of yeast Rnr1p. We introduced several mutations into RNR1-loop2 that resulted in changes in the amino acid sequence of the protein. Each of the rnr1-loop2 mutation strains obtained had different levels of individual dNTPs relative to the others. Interestingly, all of the imbalanced dNTP concentrations led to increased mutation rates, but these mutagenic imbalances did not activate the S-phase checkpoint unless one or several dNTPs were present at concentrations that were too low to sustain DNA replication. We were able to use these mutant yeast strains to successfully correlate amino acid substitutions within loop2 of Rnr1p to specific ratios of dNTP concentrations in the cells. We also demonstrated that specific imbalances between the individual dNTP levels result in unique mutation spectra. These mutation spectra suggest that the mutagenesis that results from imbalanced dNTP pools is due to a decrease in fidelity of the replicative DNA polymerases at specific DNA sequences where they are more likely to make a mistake. The mutant rnr1-loop2 strains that we have created with defined dNTP pool imbalances will be of great value for in vivo studies of polymerase fidelity, translesion synthesis by specialized DNA polymerases, and lesion recognition by the DNA repair machinery.

Master of Science / Division of Biology / Nicholas A. Wallace / Cancer is not a single story, but rather numerous often interwoven tales, each with its own characters and progression. In the case of human papillomavirus (HPV) induced cervical cancer (CaCx), the narrative is about the relationship between virus and host, with the consequences of evolution’s shortsightedness driving the plot. Along with the increased proliferative state characteristic of cancer, cells experience frequent, inaccurate replication and replication stresses (ex. DNA damage and nucleotide starvation). To prevent replication fork stall and collapse generated by these stresses, the cell employs translesion synthesis (TLS). Notably, most of the genes in this pathway are upregulated in CaCx; however, the key protein polymerase eta is not. We have observed that upregulation in this pathway is complicated. It occurs at numerous levels, increasing both mRNA and protein abundance. This research further dissects how TLS upregulation occurs. Data shows that in CaCx-derived cell lines, the stability of some TLS proteins is increased, while the stability of other TLS proteins is unchanged. The increased proliferation, typical of these cell lines, cannot account for the enhanced stability. Despite increased TLS protein stability, these cells fail to adequately activate TLS increasing the risk of DNA damage. Genomic instability is a driving factor in HPV genome integration that prevents viral propagation and leads to cell transformation. It also raises mutagenesis rates, likely creating a selective pressure for tolerating failed TLS. The elevated mutation rate known to be associated with failed TLS could also provide a mechanism for acquired resistance to the drugs commonly used to treat CaCx. Changes in protein abundance are routinely used as biomarkers that can lead to the improved outcomes associated with early cancer detection. Elevated TLS protein could be leveraged to ensure cervical cancers are detected during Stage 1, when the 5-year survival rate is 80-90%, rather than at Stage IV, when the rate dips to around 15%.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20559号 / 医博第4244号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高田 穣, 教授 萩原 正敏, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<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

To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA polymerases involved in DNA replication and/or DNA repair. The two replicative DNA polymerases, DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA polymerase epsilon participates in DNA transactions. To investigate whether Pol epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand polymerase, leaving Pol epsilon to carry out some other function. The structure of Pol epsilon was determined by cryo-electron microscopy, to a resolution of ~20 Å. Pol epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol epsilon depends on the interaction between the tail domain and double-stranded DNA. As a replicative DNA polymerase, Pol epsilon encounters different lesions in DNA. It was shown that Pol epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template. Yeast cells treated with a DNA damaging agent and devoid of all TLS polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels. In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA polymerase. The replicative DNA polymerase Pol epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.

DNA-damage tolerance (DDT) in yeast is composed of two parallel pathways and mediated by sequential ubiquitination of proliferating cell nuclear antigen (PCNA). While monoubiquitination of PCNA promotes translesion synthesis (TLS), which is dependent on low fidelity polymerase ζ (Pol ζ) composed of a catalytic subunit Rev3 and a regulatory subunit Rev7, polyubiquitination of PCNA by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Inactivation of these two pathways results in a synergistic effect on DNA-damage responses; however, this two-branch DDT model has not been reported in any multicellular organisms. In order to examine whether Arabidopsis thaliana possesses a two-branch DDT system, rad5a rev3 double mutant plants were created and compared with the corresponding single mutants. Arabidopsis rad5a and rev3 mutations are indeed synergistic with respect to growth inhibition induced by replication-blocking lesions, suggesting that AtRAD5a and AtREV3 are required for error-free and TLS branches of DDT, respectively. Unexpectedly this study reveals three modes of genetic interactions in response to different types of DNA damage, indicating that plant RAD5 and REV3are also involved in DNA damage responses independent of DDT. By comparing with yeast cells, it is apparent that plant TLS is a more frequently utilized means of lesion bypass than error-free DDT. In addition, it was also observed that treatments with the DNA damaging agent methylmethanesulfonate increased the nuclear ploidy level in the double mutant plants.

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases form a complex at the stalled replication fork called the mutasome. This complex is structurally organized by the replication accessory factor PCNA and the non-classical DNA polymerase Rev1. One of the non-classical DNA polymerases within the mutasome then catalyzes replication through the damage. Each non-classical DNA polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical DNA polymerase within the mutasome is chosen to bypass the damage. In this thesis, I discuss how the most appropriate polymerase is chosen. In so doing, I examine the components of the mutasome; the structural motifs that mediate the protein interactions in the mutasome; the methods used to study translesion synthesis; the definition of a cognate lesion; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome. Taken together, this thesis provides and inclusive review of the current state of what is known about translesion synthesis with conclusions at its end suggesting what major questions remain and ideas of how to answer them.

DNA damage blocks replication by classical DNA polymerases, those that replicate nondamaged DNA during normal DNA replication and repair, by altering the geometry of the DNA. Consequently, translesion synthesis, the replication of damaged DNA, is catalyzed by non-classical DNA polymerases, which are capable of accommodating the inherent distorted geometry of damaged DNA. The yeast Rev1 protein (Rev1p) specifically catalyzes the incorporation of cytosine opposite template guanine and several types of DNA damage utilizing a unique mechanism of nucleotide selection whereby the sidechain of Arg-324 acts as the template by forming hydrogen bonds with the incoming cytosine. To better understand the impact of this protein-template-directed mechanism on nucleotide incorporation, I carried out pre-steady-state kinetic studies with Rev1p. Interestingly, I found that Rev1p's specificity for incorporating cytosine is achieved solely at the initial nucleotide-binding step. In this respect, Rev1p differs from all previously investigated DNA polymerases. Based on these findings and on structures of another enzyme, MutM, I suggest possible structures for complexes of Rev1p with the other incoming nucleotides. DNA polymerase ζ, encoded by the REV3 gene, functions in the error-prone replication of a wide range of DNA lesions by extending from nucleotides incorporated opposite template lesions by other polymerases. Here I describe genetic and biochemical studies of five yeast DNA polymerase ζ mutant proteins. Four mutant proteins do not complement the rev3Δ mutation, and these proteins have significantly reduced or no polymerase activity relative to the wild-type protein. However, the K1061A protein partially complements the rev3Δ mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased ability to distinguish between correct and incorrect substrates (increased fidelity and decreased misextension ability). These findings have important implications for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions. Additionally, I genetically characterized 21 mutant proteins, which may also affect the substrate specificity of this enzyme. The P962L, L1054A, T1063A, and G1215A mutant proteins were partially capable of complementing the rev3Δ mutation and are candidates for biochemical characterization, as they may have altered substrate specificity.