RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

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18879号 / 医博第3990号 / 新制||医||1008(附属図書館) / 31830 / 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 小松 賢志 / 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(人間・環境学) / 甲第19068号 / 人博第721号 / 新制||人||173(附属図書館) / 26||人博||721(吉田南総合図書館) / 32019 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 小松 賢志, 教授 宮下 英明, 准教授 三浦 智行 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

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

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24272号 / 生博第486号 / 新制||生||64(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松本 智裕, 教授 高田 穣, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / 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