京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22373号 / 医博第4614号 / 新制||医||1043(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 武田 俊一, 教授 小川 誠司, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Kei Ishii, Masamichi Ishiai, Hiroko Morimoto, Mito Kanatsu-Shinohara, Ohtsura Niwa, Minoru Takata, Takashi Shinohara, The Trp53-Trp53inp1-Tnfrsf10b Pathway Regulates the Radiation Response of Mouse Spermatogonial Stem Cells, Stem Cell Reports, Volume 3, Issue 4, 14 October 2014, Pages 676-689, ISSN 2213-6711 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18685号 / 医博第3957号 / 新制||医||1007(附属図書館) / 31618 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 藤田 潤, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chromosomes are very dynamic structures that are constantly undergoing physical changes necessary for cell survival. Studies in yeast and metazoans have shown that chromosomal loci exhibit large-scale changes in mobility in response to DNA double-strand breaks (DSBs). If left unrepaired, DSBs can lead to disease and even cell death. One of the predominant cellular pathways utilized to repair DSBs is homologous recombination (HR). DSB repair via HR requires a homologous DNA template to recover the missing genetic information lost at the break site. Our lab proposes that increased chromosome mobility (ICM) facilitates recombination by helping a broken chromosome successfully find its homolog. In support of this view, ICM is under the genetic control of the HR machinery and requires activation of the DNA damage checkpoint response. However, there is currently no consensus on the precise functional role of ICM in HR. In Chapter 1, I describe in detail the known steps of DSB repair via the HR pathway, and discuss some of the important advancements made in the field of cell biology that has helped shape our understanding of HR. I highlight the use of in vivo cell imaging and fluorescently labeled DNA repair proteins during the study of HR. Additionally, I discuss some of the first studies that examined chromosome dynamics within the nucleus in live cells. Lastly, I describe the phenomenon of increased chromosome mobility and expand upon why it needs to be studied further. In Chapter 2, I present in detail our method for measuring the pairing of DNA loci during HR at a site-specific DSB in Saccharomyces cerevisiae. This method utilizes live cell imaging and a chromosome tagging system in diploid yeast to visualize homologous chromosomes during HR-mediated repair. Using this method, we demonstrate that in wild type (WT) cells, homologous chromosomes come together, repair and then move apart after repair is complete. Importantly, the kinetics we observe in the pairing of homologous chromosomes match the kinetics of site-specific DSB formation and the subsequent gene conversion of that site. In Chapter 3, I describe our study that elucidates the relationship between ICM and multiple HR steps. We find a tight temporal correlation between the recruitment of the recombination proteins, ICM, the physical pairing of homologous loci, and gene conversion. Importantly, we can shift the timing of ICM by altering the initiation of DNA end resection - an early step in the HR process. Our data highlight the importance of DNA end resection as a vital precursor to ICM and demonstrate a strong temporal linkage between ICM and HR. Taken together our data support the claim that ICM is essential to HR and mechanistically involved in the process of DNA repair. In Chapter 4, we explore chromosome mobility in response to different forms of DNA damage such as spontaneous DSBs, collapsed replication forks, and ionizing radiation (IR). We find that spontaneous DSBs and collapsed replication forks do not induce a change in chromosome mobility. However, exposure to ionizing radiation results in a robust increase in global chromosome mobility that is dependent on activation of the DNA damage checkpoint. Overall, these findings demonstrate how ICM is tightly regulated and highly dependent on the circumstances surrounding the formation of the DSB. Lastly, in Chapter 5, I summarize all of my findings and discuss how they relate to one another with respect to the linkage between ICM and HR. I also provide a perspective on future experiments needed to advance the field.

No description available.

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progressions to allow sufficient time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses to DNA damage induced by UV irradiation. However, how these two processes are coordinated in the response is poorly understood. Here we showed that the essential NER factor XPA (xeroderma pigmentosum group A) underwent nuclear accumulation upon UV irradiation, and strikingly, such an event occurred in an ATR (Ataxia-Telangiectasia mutated and RAD3-related)-dependent manner. Either treatment of cells with ATR kinase inhibitors or transfection of cells with small interfering RNA targeting ATR compromised the UV-induced XPA nuclear translocation. Consistently, the ATR-deficient cells displayed no substantial XPA nuclear translocation while the translocation remained intact in ATM (Ataxia-Telangiectasia mutated)-deficient cells in response to UV irradiation. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggested that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23607号 / 医博第4794号 / 新制||医||1055(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 溝脇 尚志, 教授 木村 剛, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Replication Factor A (RFA) is an essential heterotrimeric single-stranded DNA (ssDNA) binding complex, comprised of Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae. RFA is required for DNA replication, repair, recombination, and cell cycle regulation. RFA acts as a sensor of ssDNA, a common intermediate of these processes, and coordinates these processes through recruitment of proteins. For example, during the DNA damage response (DDR), RFA-coated ssDNA is necessary for the recruitment and activation of the sensor kinase Mec1. Additional checkpoint proteins, also recruited by RFA, are necessary for the downstream recruitment and activation of the effector kinase Rad53 that ultimately leads to cell cycle arrest. Thus, RFA acts as a bridge to recruit the proteins required for checkpoint regulation in response to DNA damage. Importantly, cell cycle resumption is contingent on Rad53 deactivation. There are two known scenarios in which Rad53 is deactivated: (1) checkpoint recovery, in which cells resume the cell cycle after DNA repair or (2) checkpoint adaptation, in which cells proceed with the cell cycle despite the continued presence of irreparable DNA damage. Previous work has demonstrated that cells undergoing checkpoint adaptation display late Rfa2 N-terminal (NT) phosphorylation that is correlated with the inactivation (dephosphorylation) of Rad53. Additionally, the use of rfa2 NT mutations consistently demonstrate that a negatively charged NT promotes adaptation in all adaptation-deficient strain backgrounds investigated. Interestingly, Rfa2 NT phosphorylation also occurs early during meiosis. This work demonstrates that: (1) Rfa1-DBD-F participates in protein-protein interactions that are sensitive to DNA damage, (2) Rfa2 phosphorylation increases the DNA damage sensitivity of mutants with deficient DNA damage checkpoints, (3) the Rfa2 NT is required for proper progression through meiosis that appears to be unrelated to RFA functions in replication or DNA repair by homologous recombination (HR), and (4) Rfa2 phosphorylation may regulate Mec1 checkpoint signaling during the DDR to control checkpoint exit and cell cycle resumption. A mechanism is proposed that considers both Rfa1 DBD-F and the Rfa2 NT involvement to initiate HR repair that essentially allows for the continuation of the cell cycle by the delocalization of Mec1.

Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.

Maintenance of genomic integrity is essential for the survival of an organism and its ability to pass genetic information to its progeny. However, DNA is constantly exposed to exogenous and endogenous sources of damage, which demands cells to possess DNA repair mechanisms. Of the many forms of DNA damage, double-strand breaks (DSBs) are particularly cytotoxic DNA lesions that cause genome instability and cell lethality, but also provide opportunities to manipulate the genome via repair. One of the major DSB repair pathways shared between single-celled yeast and humans is homologous recombination (HR). HR is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/Nbs1 (MRX in yeast, MRN in mammals) complex. The MRX complex has a multitude of functions such as damage sensing, adduct removal from DSB ends, and end tethering – a process to maintain the two ends of a DSB in close proximity. The role of the MRX complex has been uncovered by studying the repair of DSBs generated from meganucleases such as HO and I-SceI. However, it is unclear if this knowledge translates to the repair of DSBs from genome editing nucleases such as TALEN and CRISPR-Cas9 (Cas9), as these nucleases create DSBs with different end polarities. While the repair efficiencies and outcomes of TALEN and Cas9 are actively studied, less is known about the earlier stages of repair. The objective of this thesis is to examine the role of the MRX complex in repair processes at both ends of a DSB after cleavage with I-SceI, TALEN, and Cas9 in vivo using the model organism Saccharomyces cerevisiae. In Chapter 1, I describe the importance of DSB repair, a summary of HR and its sub-pathways, the functions of the MRX complex, and properties of I-SceI, TALEN, and Cas9. The materials and methods used in this thesis are detailed in Chapter 2. The work described in Chapter 3 focuses on end tethering and recruitment of downstream repair proteins in haploid cells. I find that DSB ends from the three nucleases all depend on the MRX complex for end tethering, and that initial end polarity does not affect tethering. DSBs created by Cas9 show greater dependence on the Mre11 nuclease of the MRX complex for Rad52 recruitment compared to DSBs from I-SceI and TALEN. Despite Mre11-dependent end processing and Rad52 recruitment at Cas9-induced DSBs, Cas9 stays bound to one DNA end after cleavage, irrespective of the MRX complex. These results suggest that Mre11 exonuclease activity required for adduct removal from DSB ends is not critical for Rad52 recruitment, and that Mre11 endonuclease activity may be driving processing of Cas9-bound DSBs. I also find that MRX tethers DSB ends even after Rad52 recruitment, and unexpectedly, untethered ends are processed asymmetrically in the absence of MRX for all three nucleases. In Chapter 4, I explore the interaction of DSB ends with their repair template, the intact homologous chromosome, in diploid cells. The primary goal is to monitor interhomolog contact in real time from homology search to completion of HR. Although technical limitations make it difficult to capture the entire HR program from DSB formation to repair, I show that untethered ends interact with the homolog separately in the absence of the MRX complex. Similar to haploids, diploid cells display defects in end tethering and end processing without the MRX complex. Repair outcomes of WT cells show an even distribution of G2 crossovers and non-crossovers, while pre-replication crossovers and break-induced replication are undetected. Overall, the results in this thesis provide insight into the functions of the MRX complex in repairing different DSB ends created by I-SceI, TALEN, and Cas9. In Chapter 5, I summarize all of these findings and discuss the motivation for future cell biology studies of HR.

No description available.