Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase λ (pol λ), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol λ can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol λ, we also show that the unique proline-rich region in pol λ plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol λ interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol λ. Taken together, these data strongly support that pol λ functions in DNA polymerization events during NHEJ.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19555号 / 医博第4062号 / 新制||医||1012(附属図書館) / 32591 / 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 戸井 雅和, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Small molecule disruption of RAD52 rings as a mechanism for precision medicine in BRCA deficient cancers Suppression of RAD52 causes synthetic lethality in BRCA deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA deficient cancers. How RNA transcripts coordinate DNA recombination and repair Genetic studies in yeast indicate that RNA transcripts facilitate homology-directed DNA repair in a manner that is dependent on RAD52. The molecular basis for so-called RNA-DNA repair, however, remains unknown. Using reconstitution assays, we demonstrate that RAD52 directly cooperates with RNA as a sequence-directed ribonucleoprotein complex to promote two related modes of RNA-DNA repair. In a RNA-bridging mechanism, RAD52 assembles recombinant RNA-DNA hybrids that coordinate synapsis and ligation of homologous DNA breaks. In a RNA-templated mechanism, RAD52 mediated RNA-DNA hybrids enable reverse transcription dependent RNA-to-DNA sequence transfer at DNA breaks that licenses subsequent DNA recombination. Notably, we show that both mechanisms of RNA-DNA repair are promoted by transcription of a homologous DNA template in trans. In summary, these data elucidate how RNA transcripts cooperate with RAD52 to coordinate homology-directed DNA recombination and repair in the absence of a DNA donor, and demonstrate a direct role for transcription in RNA-DNA repair. Characterization of DNA polymerase θ as a reverse transcriptase RNA-to-DNA sequence has been observed in human cells, but how the phenomena occurs remains unknown. Multiple lines of evidence suggest putative reverse transcriptase (RT) activity as a potential mechanism for how RNA sequence can alter chromosomal DNA, but the source of this RT remains unknown. Here, we have identified that the unique A-family DNA polymerase theta (Polθ) displays robust RT activity, a characteristic not found in any other human polymerase tested from the A, B, X, and Y families. We propose that Polθ may be responsible for the observed RT activity in human cells. / Biomedical Sciences

Cockayne syndrome (CS) is a rare, segmental premature aging disorder in which the majority of cases are caused by mutations in the Cockayne syndrome group B protein (CSB). CSB is a multifunctional protein implicated in DNA repair, transcription and chromatin remodeling. The results presented here demonstrate that CSB plays an important role in telomere maintenance and DSB repair. We find that CS cells accumulate telomere doublets, have increased telomere-bound TRF1, decreased TERRA levels and a defect in telomerase-dependent telomere lengthening. These results imply that CS patients may be defective in telomere maintenance. We also uncover a novel and important role of CSB in DNA DSB repair. We show that CSB facilitates HR and supresses NHEJ during S and G2 phase. We find that CSB interacts with RIF1 and is recruited by RIF1 to DSBs in S phase. At DSBs, CSB remodels the chromatin extensively, which in turn limits RIF1 recruitment and promotes BRCA1 accumulation. The chromatin remodeling activity of CSB requires not only damage-induced phosphorylation on S10 by ATM but also cell cycle-dependent phosphorylation of S158 by cyclin A-CDK2. Both modifications are needed for the intramolecular interaction of CSB N-terminal domain with its ATPase domain. This intramolecular interaction has previously been reported to regulate the ATPase activity of CSB. Taken together, these results suggest that ATM and CDK2 control of CSB to promote chromatin remodeling, which in turn inhibits RIF1 in DNA DSB repair pathway choice. / Thesis / Doctor of Philosophy (PhD)

The amino acid proline is unique – within a polypeptide chain, proline adopts either a cis or trans peptide bond conformation while all other amino acids are sterically bound primarily in the trans configuration. In proteins, the isomeric state of a single proline can have dramatic consequences on structure and function. Consequently, cis-trans interconversion confers both barrier and opportunity – on one hand, isomerization is a rate limiting step in de novo protein folding and on the other can be utilized as a post-translational regulatory switch. Peptidyl-prolyl isomerases (PPIs) are a ubiquitous superfamily that catalyzes the interconversion between conformers. Although pervasive, the functions and substrates of most PPIs are unknown. The two largest subfamilies, FKBPs and cyclophilins, are the intracellular receptors of clinically relevant immunosuppressant drugs that also show promise in the treatment of neurodegenerative disorders and cancer. Therefore, narrowing the knowledge gap has significant potential to benefit human health. FKBP25 is a high-affinity binder of the PPI inhibitor rapamycin and is one of few nuclear-localized isomerases. While it has been shown to bind DNA and associate with chromatin, its function has remained largely uncharacterized. I hypothesized that FKBP25 targets prolines in nuclear proteins to regulate chromatin-templated processes. To explore this, I performed high-throughput transcriptomic and proteomic studies followed by detailed molecular characterizations of FKBP25’s function. Here, I discover that FKBP25 is a multifunctional protein required for the maintenance of genomic stability. In Chapter 2, I characterize the unique N-terminal Basic Tilted Helical Bundle (BTHB) domain of FKBP25 as a novel dsRNA binding module that recruits FKBP25’s prolyl isomerase activity to pre-ribosomal particles in the nucleolus. In Chapter 3, I show for the first time that FKBP25 associates with the mitotic spindle apparatus and acts to stabilize the microtubule cytoskeleton. In this chapter, I also present evidence that this function influences the stress response, cell cycle, and chromosomal stability. Additionally, I characterize the regulation of FKBP25’s localization and nucleic acid binding activity throughout the cell cycle. Finally, in Chapter 4, I uncover a role for FKBP25 in the repair of DNA double-stranded breaks. Importantly, this function requires FKBP25’s catalytic activity, identifying for the first time a functional requirement for cis-trans prolyl isomerization by FKBP25. Collectively, this work identifies FBKP25 as a multifunctional protein that is required for the maintenance of genomic stability. The knowledge gained contributes to the exploration of PPIs as important drug targets. / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22700号 / 医科博第115号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19401号 / 医博第4052号 / 新制||医||1012(附属図書館) / 32426 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙田 穣, 教授 平岡 眞寛, 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

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24520号 / 医博第4962号 / 新制||医||1065(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 生田 宏一, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM