Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24946号 / 生博第508号 / 新制||生||68(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 松田 道行, 教授 松本 智裕, 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 / DGAM

A instabilidade genômica tem sido implicada como um dos principais fatores relacionados ao processo de envelhecimento. Esta é consequência do acumulo de danos no DNA em células somáticas continuamente expostas a fatores endógenos e exógenos. Um grupo de proteínas que desempenha diversos papéis na manutenção e estabilidade do genoma é formado pelas RecQ helicases, atuando em vários processos do metabolismo celular, tais como replicação do DNA, recombinação, reparo do DNA e manutenção dos telômeros. Algumas evidencias relacionam a expressão aberrante destas proteínas ao envelhecimento precoce. Com o objetivo de determinar os perfis de expressão transcricional de genes da família RecQ helicase e alguns genes envolvidos na via BER (Base excision repair), como PARP1, POL e APEX1 em células mononucleares do sangue periférico (PBMCs, do inglês Peripheral Blood Mononuclear Cells), comparamos grupos de indivíduos jovens (n = 20), idosos (n = 17) e centenários (n = 27). Além disso, foi também foi avaliado o comprimento telomérico em amostras de DNA desses indivíduos, buscando uma comparação entre os mesmos. Foi observada uma diminuição no nível de expressão transcricional do gene BLM nos grupos idoso e centenário quando comparados ao grupo jovem (p<0,05). Também foi observado uma diminuição na expressão do gene RECQL5 no grupo idoso comparado ao grupo jovem. Para os genes da via BER, foi observada uma repressão na expressão transcricional de PARP1 no grupo idoso em relação ao grupo jovem (p<0,05). Em relação ao comprimento telomérico, nossos resultados demonstraram associação entre a diminuição do comprimento telomérico e a idade. Obtivemos diferença significativa na comparação do comprimento telomérico de idosos e centenários comparados ao grupo jovem. Porém, não foi observada diferença entre os grupos idosos e centenários. Assim, nossos resultados mostram uma associação do processo de envelhecimento com a modulação de alguns genes da família RecQ helicase e participantes da via BER, e com o encurtamento telomérico. Os resultados gerados nesse trabalho são inéditos, sendo que relevantes para melhor compreensão do processo de envelhecimento. / Genomic instability plays a major role in the aging process due to the accumulation of DNA damage in somatic cells continuously exposed to endogenous and exogenous factors. A group of proteins essential in maintaining genome stability is composed by RecQ helicase, acting in several cell metabolism processes such as DNA replication, recombination, DNA repair and telomere maintenance. Some evidence related the aberrant expression of these proteins to premature aging. In order to determine the transcriptional expression profile of RecQ helicase gene family and some genes involved in the BER (Base excision repair) pathway, such as PARP1, POL and APEX1 in peripheral blood mononuclear cells (PBMCs), we compared groups of young (n = 20), elderly (n = 17) and centenarians (n = 27). Furthermore, it was also evaluated telomere length in DNA samples from these individuals. It was observed a decrease in the transcriptional expression of BLM gene in elderly and centenarians compared to the young group (p <0.05). It was also observed a decrease in expression of RECQL5 gene in the elderly compared to the younger group. For the BER genes, it was observed a transcriptional repression of PARP1 in the elderly group compared to the young group (p <0.05). Regarding the telomere length, our results demonstrated an association between reduction of telomere length and age. We obtained significant difference in comparing the telomere length of the elderly and centenarians compared to the younger group. However, no difference was observed between the elderly and centenarians groups. Thus, our results show an association of aging process with the modulation of certain genes from RecQ helicase family and participants of the BER pathway and the telomere shortening. The results generated in this study are promising, and relevant to better understanding the aging process.

A instabilidade genômica tem sido implicada como um dos principais fatores relacionados ao processo de envelhecimento. Esta é consequência do acumulo de danos no DNA em células somáticas continuamente expostas a fatores endógenos e exógenos. Um grupo de proteínas que desempenha diversos papéis na manutenção e estabilidade do genoma é formado pelas RecQ helicases, atuando em vários processos do metabolismo celular, tais como replicação do DNA, recombinação, reparo do DNA e manutenção dos telômeros. Algumas evidencias relacionam a expressão aberrante destas proteínas ao envelhecimento precoce. Com o objetivo de determinar os perfis de expressão transcricional de genes da família RecQ helicase e alguns genes envolvidos na via BER (Base excision repair), como PARP1, POL e APEX1 em células mononucleares do sangue periférico (PBMCs, do inglês Peripheral Blood Mononuclear Cells), comparamos grupos de indivíduos jovens (n = 20), idosos (n = 17) e centenários (n = 27). Além disso, foi também foi avaliado o comprimento telomérico em amostras de DNA desses indivíduos, buscando uma comparação entre os mesmos. Foi observada uma diminuição no nível de expressão transcricional do gene BLM nos grupos idoso e centenário quando comparados ao grupo jovem (p<0,05). Também foi observado uma diminuição na expressão do gene RECQL5 no grupo idoso comparado ao grupo jovem. Para os genes da via BER, foi observada uma repressão na expressão transcricional de PARP1 no grupo idoso em relação ao grupo jovem (p<0,05). Em relação ao comprimento telomérico, nossos resultados demonstraram associação entre a diminuição do comprimento telomérico e a idade. Obtivemos diferença significativa na comparação do comprimento telomérico de idosos e centenários comparados ao grupo jovem. Porém, não foi observada diferença entre os grupos idosos e centenários. Assim, nossos resultados mostram uma associação do processo de envelhecimento com a modulação de alguns genes da família RecQ helicase e participantes da via BER, e com o encurtamento telomérico. Os resultados gerados nesse trabalho são inéditos, sendo que relevantes para melhor compreensão do processo de envelhecimento. / Genomic instability plays a major role in the aging process due to the accumulation of DNA damage in somatic cells continuously exposed to endogenous and exogenous factors. A group of proteins essential in maintaining genome stability is composed by RecQ helicase, acting in several cell metabolism processes such as DNA replication, recombination, DNA repair and telomere maintenance. Some evidence related the aberrant expression of these proteins to premature aging. In order to determine the transcriptional expression profile of RecQ helicase gene family and some genes involved in the BER (Base excision repair) pathway, such as PARP1, POL and APEX1 in peripheral blood mononuclear cells (PBMCs), we compared groups of young (n = 20), elderly (n = 17) and centenarians (n = 27). Furthermore, it was also evaluated telomere length in DNA samples from these individuals. It was observed a decrease in the transcriptional expression of BLM gene in elderly and centenarians compared to the young group (p <0.05). It was also observed a decrease in expression of RECQL5 gene in the elderly compared to the younger group. For the BER genes, it was observed a transcriptional repression of PARP1 in the elderly group compared to the young group (p <0.05). Regarding the telomere length, our results demonstrated an association between reduction of telomere length and age. We obtained significant difference in comparing the telomere length of the elderly and centenarians compared to the younger group. However, no difference was observed between the elderly and centenarians groups. Thus, our results show an association of aging process with the modulation of certain genes from RecQ helicase family and participants of the BER pathway and the telomere shortening. The results generated in this study are promising, and relevant to better understanding the aging process.

Dans les cellules, le déroulement de l'ADN double-brin est catalysé par une famille de protéines appelées hélicases. Ces protéines sont présentes chez tous les organismes des virus jusqu'à l'homme. Parmi ces hélicases, celles de la famille RecQ jouent un rôle essentiel dans le métabolisme de l'ADN en facilitant de nombreux processus cellulaires tels que la réplication, la réparation, la recombinaison, la transcription et la maintenance des télomères. Chez l'homme, il existe cinq membres de la famille RecQ identifiés comme RECQ1, BLM, WRN, RECQ4 et RECQ5. Les mutations dans BLM, WRN et RECQ4 sont associées à une prédisposition au cancer. En plus du domaine hélicase très conservé et contenant sept motifs bien distincts, la plupart des hélicases de la famille RecQ possèdent également un domaine RecQ C-terminal (RecQ-Ct) et un domaine hélicase RNase D (HRDC). Au cours de ce travail, nous nous concentrons sur les mécanismes intrafonctionnels de certains membres de la famille RecQ des hélicases. Tout d'abord, nous avons utilisé deux isoformes naturels de l'hélicase RECQ5 humain comme modèle pour étudier la modulation fonctionnelle du domaine hélicase avec le doigt de zinc. Ici, nous montrons que la variante tronquée RECQ5α de l'hélicase RECQ5β issue d'un épissage alternatif et composée uniquement du domaine hélicase ne possède ni l'activité ATPase ni l'activité de déroulement de l'ADN. A l'inverse, et ce de matière étonnante, cette protéine est dotée d'une forte activité de réhybridation du brin déroulé. Les mesures quantitatives indiquent que l'amélioration de l'affinité de la protéine pour l'ADN que lui confère le doigt de zinc est à l'origine de ses activités ATPase et hélicase. Le plus important est que l'on constate que le doigt de zinc est capable d'agir comme un facteur moléculaire à même de supprimer l'activité de re-synthèse du brin déroulé par le domaine hélicase et de déclencher l'activité de déroulement d'ADN à travers une modulation de la fixation à l'ADN. Ensuite, nous avons analysé les propriétés biochimiques de deux isoformes de l'hélicase RecQ de Bacillus subtilis : SubL et SubS. Parmi elles, SubS ne dispose pas du domaine HRDC. Nos études montrent que le domaine HRDC est crucial pour Bacillus subtilis RecQ hélicases dans la résolution des intermédiaires de réplication et / ou de réparation de l'ADN tels que les jonctions de Holliday et la jonction de kappa. Les activités ATPase, hélicase et l'activité de rehybridation du brin déroulé sont plus importantes en présence du domaine HRDC. Ces résultats nous permettent de spéculer sur l'importance du domaine HRDC des activités de la famille de RecQ hélicase. Nous avons découvert que dans la famille RecQ, le 12 domaine HRDC peut augmenter les activités ATPases et hélicases. De manière intéressante, le domaine HRDC de Bacillus subtilis joue un rôle critique dans la résolution des intermédiaires de réplication ou de réparation de l'ADN et des jonctions de Holliday. Nous suggérons l'hypothèse que le domaine HRDC des hélicases RecQ participe à exposer leurs fonctions dans le processus de réparation de l'ADN. Dans la dernière partie, nous nous sommes intéressés à l'existence et au rôle du doigt d'arginine dans la protéine BLM. Ces études ont été menées afin de démontrer son rôle dans l'hydrolyse d'ATP et dans la conversion en mouvement mécanique permettant à la protéine de progresser le long de l'ADN. Nos études démontrent que le résidu R982, situé à proximité du γ-phosphate de l'ATP, fonctionne comme un doigt d'arginine dans la protéine BLM. Nos conclusions indiquent en outre que ce doigt d'arginine interagit avec d'autres motifs conservés situés autour du γ-phosphate des nucléotides et qu'ils effectuent ensemble les fonctions enzymatiques au sein d'un réseau complexe.

Genome instability is a hallmark of human cancers. Patients with Bloom’s syndrome, a rare chromosome breakage syndrome caused by inactivation of the RecQ helicase BLM, result in phenotypes associated with accelerated aging and develop cancer at a very young age. Patients with Bloom’s syndrome exhibit hyper-recombination, but the role of BLM and increased genomic instability is not fully characterized. Sgs1, the only member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is known to act both in early and late stages of homology-dependent repair of DNA damage. Exo1, a 5′–3′ exonuclease, first discovered to play a role in mismatch repair has been shown to participate in parallel to Sgs1 in processing the ends of DNA double-strand breaks, an early step of homology-mediated repair. Here we have characterized the genetic interaction of SGS1 and EXO1 with other repair factors in homology-mediated repair as well as DNA damage checkpoints, and characterize the role of post-translational modifications, and protein-protein interactions in regulating their function in response to DNA damage. In S. cerevisiae cells lacking Sgs1, spontaneous translocations arise by homologous recombination in small regions of homology between three non-allelic, but related sequences in the genes CAN1, LYP1, and ALP1. We have found that these translocation events are inhibited if cells lack Mec1/ATR kinase while Tel1/ATM acts as a suppressor, and that they are dependent on Rad59, a protein known to function as one of two sub-pathways of Rad52 homology-directed repair. Through a candidate screen of other DNA metabolic factors, we identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1∆ mutant. The Exo1 enzymatic domain is located in the N-terminus while the C-terminus harbors mismatch repair protein binding sites as well as phosphorylation sites known to modulate its enzymatic function at uncapped telomeres. We have determined that the C-terminus is dispensable for Exo1’s roles in resistance to DNA-damaging agents and suppressing mutations and chromosomal rearrangements. Exo1 has been identified as a component of the error-free DNA damage tolerance pathway of template switching. Exo1 promotes template switching by extending the single strand gap behind stalled replication forks. Here, we show that the dysregulation of the phosphorylation of the C-terminus of Exo1 is detrimental in cells under replication stress whereas loss of Exo1 suppresses under the same conditions, suggesting that Exo1 function is tightly regulated by both phosphorylation and dephosphorylation and is important in properly modulating the DNA damage response at stalled forks. It has previously been shown that the strand exchange factor Rad51 binds to the C-terminus of Sgs1 although the significance of this physical interaction has yet to be determined. To elucidate the function of the physical interaction of Sgs1 and Rad51, we have generated a separation of function allele of SGS1 with a single amino acid change (sgs1-FD) that ablates the physical interaction with Rad51. Alone, the loss of the interaction of Sgs1 and Rad51 in our sgs1-FD mutant did not cause any of the defects in response to DNA damaging agents or genome rearrangements that are observed in the sgs1 deletion mutant. However, when we assessed the sgs1-FD mutant in combination with the loss of Sae2, Mre11, Exo1, Srs2, Rrm3, and Pol32 we observed genetic interactions that distinguish the sgs1-FD mutant from the sgs1∆mutant. Negative and positive genetic interactions with SAE2, MRE11, EXO1, SRS2, RRM3, and POL32 suggest the role of the physical interaction of Sgs1 and Rad51 is in promoting homology-mediated repair possibly by competing with single-strand binding protein RPA for single-stranded DNA to promote Rad51 filament formation. Together, these studies characterize additional roles for domains of Sgs1 and Exo1 that are not entirely understood as well as their roles in combination with DNA damage checkpoints, and repair pathways that are necessary for maintaining genome stability.

Double-strand breaks and stalled forks arise when the replication machinery encounters damage from exogenous sources like DNA damaging agents or ionizing radiation, and require specific DNA helicases to resolve these structures. Sgs1 of Saccharomyces cerevisiae is a member of the RecQ family of DNA helicases and has a role in DNA repair and recombination. The RecQ family includes human genes BLM, WRN, RECQL4, RECQL1, and RECQL5. Mutations in BLM, WRN, and RECQL4 result in genetic disorders characterized by developmental abnormalities and a predisposition to cancer. All RecQ helicases have common features including a helicase domain, an RQC domain, and a HRDC domain. In order to elucidate the role of these domains and to identify additional regions in Sgs1 that are required for the maintenance of genome integrity, a series of systematic truncations to the C terminus of Sgs1 were created. We found that ablating the HRDC domain does not cause an increase in accumulating gross chromosomal rearrangements (GCRs). But deleting the RQC domain and leaving the helicase domain intact resulted in a rate similar to that of a helicase-defective mutant. Additionally, we exposed these truncation mutants to HU and MMS and demonstrated that losing up to 200 amino acids from the C terminus did not increase sensitivity to HU or MMS, whereas losing 300 amino acids or more led to sensitivity similar to that of an sgs1∆ cell. These results suggest that the RQC domain, believed to mediate protein-protein interactions and required for DNA recognition, is important for Sgs1’s role in suppressing GCRs and sensitivity to HU and MMS, whereas the HRDC domain that is important for DNA binding is not necessary. RecQL5 is a RecQ-like helicase that is distinct from the other members through its three different isoforms, RecQL5α, RecQL5β, and RecQL5ɣ. It has a helicase domain and an RQC domain, but lacks the HRDC domain that other RecQ-like helicases possess. In contrast to Blm, Wrn, and RecQL4, no human disorder has been associated with defects in RecQL5. For this reason the role of RecQL5 in the cell has remained largely unknown. To try to elucidate the pathways RecQL5 may be involved in we performed a yeast two hybrid to identify RecQL5-interacting proteins. We found that RecQL5 interacts with Hlp2, an ATP-dependent RNA helicase, and Ube2I, a SUMO-conjugating enzyme. These novel interactions shed light on a potential role of RecQL5 in the cell as a transcriptional regulator. Saccharomyces cerevisiae, Rrm3, is a 5’-3’ DNA helicase that is part of the Pif1 family of DNA helicases and is conserved from yeast to humans. It was initially discovered as a suppressor of recombination between tandem arrays and ribosomal DNA (rDNA) repeats. In its absence there are increased rates of extra-chromosomal rDNA circles, and cells accumulate X-shaped intermediates at stalled forks. Rrm3 may be involved in displacing DNA-protein blocks and unwinding DNA to facilitate fork progression. We used stable isotope labeling by amino acids in cell culture (SILAC)- based quantitative mass spectrometry in order to determine proteins that deal with the stalled fork in the absence of Rrm3. We found that in the absence of Rrm3 and increased replication fork pausing, there is a requirement for the error-free DNA damage bypass factor Rad5 and the homologous recombination factor Rdh54 for fork recovery. We also report a novel role for Rrm3 in controlling DNA synthesis upon exposure to replication stress and that this requirement is due to interaction with Orc5, a subunit of the origin recognition complex. Interaction of Orc5 was found to be located within a 26-residue region in the unstructured N-terminal tail of Rrm3 and loss of this interaction resulted in lethality with cells devoid of the replication checkpoint mediator Mrc1, and DNA damage sensitivity with cells lacking Tof1. In this study we describe two independent roles of Rrm3, a helicase-dependent role that requires Rad5 and Rdh54 for fork recovery, and a helicase-independent role that requires Orc5 interaction to control DNA synthesis. Our data provides novel insight into the role of DNA helicases and their role in protecting the genome. Through yeast genetics it was possible to determine the importance of the C terminus of Sgs1 and elucidate new RecQL5 interacting partners that shed light onto roles for RecQL5 distinct from other RecQ like helicases. Quantitative mass spectrometry allowed us to take on a more global view of the cell and determine how it responds to replication fork pausing in the absence of Rrm3. Using both proteomics and yeast genetics we were able to better understand how these DNA helicases contribute to maintaining genome stability.

No description available.

Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition. WS cells show increased genomic instability and are hypersensitive to DNA-damaging agents. WS is caused by mutations of the WRN gene. WRN protein is a member of RecQ DNA helicase family. In addition to a conserved 3’–5’ helicase activity, the WRN protein contains unique 3’–5’ exonuclease activity. WRN recognizes specific DNA structures as substrates that are intermediates of DNA metabolism. WRN physically and functionally interacts with many other proteins that function in telomere maintenance, DNA replication, and DNA repair. The function of WRN is regulated by post–translational modifications that include phosphorylation, acetylation, and sumoylation. SIRT1 is a NAD-dependent histone deacetylase (HDAC) that deacetylates histones and a numbers of cellular proteins. SIRT1 regulates the functions of many proteins, which are important for apoptosis, cell proliferation, cellular metabolism, and DNA repair. SIRT1 is also regulated by other proteins or molecules from different levels to activate or inhibit its deacetylase activity. In this study, we found that SIRT1 interacts with and deacetylates WRN. We further identified the major acetylation sites at six lysine residues of the WRN protein and made a WRN acetylation mutant for functional analysis. We found that WRN acetylation increases its protein stability. Deacetylation of WRN by SIRT1 reverses this effect. CREB-binding protein (CBP) dramatically increased the half-life of wild-type WRN, while this increase was abrogated with the WRN acetylation mutant. We further found that WRN stability is regulated by the ubiquitination pathway, and that WRN acetylation by CBP dramatically reduces its ubiquitination level. We also found that acetylation of WRN decreases its helicase and exonuclease activities, and that SIRT1 reverses this effect. Acetylation of WRN alters its nuclear distribution. Down-regulation of SIRT1 increases WRN acetylation level and prevents WRN protein translocating back to nucleolus after DNA damage. Importantly, we found that WRN protein is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells that were stably expressing WRN acetylation mutant display significantly higher sensitivity to MMC than the cells expressing wild-type WRN. Taken together, these data demonstrated that acetylation pathway plays an important role in regulating WRN function in response to DNA damage. A model has been proposed based on our discoveries.

Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired. Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial. Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing. Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V. We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-xhypomorph to completely fail to slow. We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were. Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing. Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.