This thesis describes the establishment of a precise gene targeting methodology in the silkworm Bombyx mori by technologies based on engineered endonucleases. Two classes of engineered endonucleases, ZFNs and full length TALENs were used for creating DSBs at specified sites in the colour marker genes (BmBlos2 and Bmwh3). Direct embryo microinjection of engineered nucleases mRNA were performed and let the nuclease proteins to disrupt the functions of these marker genes by creating DSBs and inducing error prone NHEJ mechanism. These experiments showed that both ZFNs and TALENs could be used for targeted gene disruption in silkworms.

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.

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.

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

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.

DNA double-strand breaks (DSB) with chemically modified end-termini are the most significant lesions resulting from radio/chemotherapeutic intervention of cancer and non homologous end-joining (NHEJ) factor Artemis nuclease has been implicated in the repair of such breaks. To examine whether the resolution of terminally blocked DNA DSBs is the biologically relevant function of Artemis, Artemis deficient fibroblasts were stably complemented with wild type or an endonuclease deficient D165N mutant Artemis. Physiological levels of wild type (WT) Artemis completely restored DSB repair proficiency and resistance to γ-radiation, bleomycin, and neocarzinostatin. Cells expressing the D165N mutants remained as chemo/radiosensitive and as repair deficient as parental cells, with persistent γ H2AX and 53BP1 foci that increased in size 6-18 hour post irradiation. These persistent foci co-localized with DNA double strand break repair factor Mre11 and also with promyelocytic leukemia protein (PML). Further, in vitro studies have revealed that DNA-PK dependent Artemis endonucleolytic activity may play a role in the repair of commonly found oxidative base damage; 8-oxoguanine (8-oxoG), a hallmark of complex DSBs. However, majority of DNA DSBs are repaired in an Artemis independent manner, and recently discovered, DNA end-specific nuclease, Metnase is a candidate enzyme for repair of such breaks. To study the role of Metnase in resolution of 3ʹ-blocked termini, several substrates mimicking such breaks were constructed. A 3ʹ-phosphoglycolate moiety on longer overhangs (4 and 6 bases) altered specificity and stimulated Metnase-mediated cleavage of the terminal 3 nucleotides. However, an 8-oxoG residue at the single-strand/double-strand border did not affect specificity or extent of cleavage. Metnase preferentially cleaved ssDNA-overhang of a partially duplex substrate, and the cleavage increased with increase in length of 3ʹ-overhangs. A D483A mutation in Metnase completely abrogated Metnase cleavage activity towards DNA ends. These results suggest that Metnase may resolve oxidatively damaged DNA ends to facilitate repair while Artemis is required for the resolution of more complex DNA DSBs that persist for longer times and are not amenable to repair by other NHEJ factors.

The integrity of the genome relies on the maintenance of chromosomes, the structural embodiment of the genetic material. Disruption of chromosome replication can lead to extensive genomic rearrangements, spanning kilobase (Kb) to megabase (Mb) regions. Some chromosome rearrangements are inherently dynamic, beginning as a single unstable rearrangement from which multiple rearrangements emerge. The rare formation and transient behavior of unstable chromosomes renders their study challenging. Here I characterize the genetic ontogeny of unstable chromosomes in a budding yeast model, from initial replication error to unstable chromosome formation to their resolution. I find that the initial error often arises in or near the telomere and frequently forms unstable chromosomes that later resolve to an internal "collection site" in the middle of the chromosome. The initial telomere-proximal unstable chromosome is increased in cells mutant for telomerase, the Tel1 checkpoint kinase and even the Rad9 checkpoint protein, with no known telomere-specific function. Defects in Tel1 and the Rrm3 DNA helicase, or the Tel1-MRX complex and 9-1-1 checkpoint clamp, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. I performed a candidate genetic screen of instability in telomere maintenance and DNA damage response (DDR) proteins to characterize the interplay of pathways regulating senescence and genomic instability. Collectively, my results suggest that unstable chromosomes form in or near damaged telomeres, independently of end degradation (Exo1-independent), by either nonhomologous end joining (partially Lig4-dependent) or by faulty template switch during replication (Lig4- and Rad52-independent). The telomere-proximal unstable chromosomes then rearrange further to the middle of the chromosome. These results implicate telomere replication errors as a common source of widespread genomic changes and make substantial progress to our understanding of the initiation and fate of unstable chromosomes in the eukaryotic genome.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.