Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

The Role of TvTrxR in Drug Resistance and Characterization of TvRad51 in Homologous Recombination in Trichomonas vaginalis Abstract By Melissa Hopper University of the Pacific 2016 In recent years, prevalence of metronidazole-resistant cases of Trichomonas vaginalis has been on the rise. With nearly 10% of strains resistant to metronidazole, new treatments to combat this parasite have become a necessity. FDA-approved drug screens have identified the compound, auranofin, as an effective agent against similar protozoans. The mechanism of inhibition by auranofin has been found to proceed through inhibition of the thioredoxin-based anti-oxidant pathway, targeting the enzyme thioredoxin reductase (TrxR). In this study, auranofin was found to be an effective inhibitor of T. vaginalis TrxR activity. Auranofin was also found to be an effective inhibitor of several trichomonad strains in culture, exhibiting IC50 values comparable to metronidazole. These studies indicate that auranofin is a promising agent for treatment of trichomoniasis. Another aspect of T. vaginalis biology addressed in this study is the ability of T. vaginalis to carry out homologous recombination (HR), a process used to repair double-stranded breaks in DNA. The protein radiation sensitive protein 51 (Rad51) plays a crucial role in the process of HR in mitotic and meiotic recombination. In this study, experiments were carried out to elucidate the role of T. vaginalis Rad51 in homologous recombination. TvRad51 was found to exhibit nuclear localization and was capable of carrying out ATP hydrolysis. Rad51 was shown to be up-regulated at the protein level in T. vaginalis in response to treatment with DNA-damaging agents. In addition, TvRad51 was capable of binding the BRC repeat region of TvBRCA2. These results indicate that T. vaginalis upregulates expression of Rad51 protein in response to certain forms of DNA damage and TvRad51 may be capable of carrying out HR mediated by different binding partners.

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 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20212号 / 理博第4297号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 准教授 小山 時隆, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21026号 / 医科博第87号 / 新制||医科||6(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 篠原 隆司, 教授 松田 道行, 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

A large number of anti-cancer chemotherapeutics target DNA topoisomerases. Etoposide is a specific topoisomerase II poison which causes reversible double strand DNA breaks. The focus of this project is to analyze the repair of DNA damage induced by etoposide.. Double strand DNA break repair is mediated by through either non-homologous end joining (NHEJ) or homologous recombination. NHEJ repairs through direct ligation of a double stranded break while homologous recombination utilizes a homologous template to recover the wild type sequence. A reporter cassette, RYDR-GFP, has been stably integrated into HeLa cells. This reporter contains an ultra-high affinity topoisomerase II cleavage site (RY) placed in the middle of a mutant GFP sequence. Flanking this sequence is a corresponding stretch of wild type GFP that is used as template to repair the break and restore gene function yielding GFP positive cells. Titrations with etoposide have shown that a logarithmic increase in drug concentration yields a corresponding increase in repair through homologous recombination (HR). This result demonstrates that topoisomerase II mediated damage is efficiently repaired by the process of HR. To examine NHEJ repair, a doxycycline inducible, stably integrated NHEJ HeLa cell reporter cassette was also evaluated. The data indicates that repair of topoisomerase II mediated DNA damage occurs more efficiently through the HR pathway. Collectively, the data suggests that tumor cells proficient in HR repair may effectively elude treatment by topoisomerase II targeting drugs.

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