Double-strand break repair and homologous recombination in Schizosaccharomyces pombe

In recent years our understanding of double strand break repair and homologous recombination in Schizosaccharomyces pombe has increased significantly, and the identification of novel pathways and genes with homologues in higher eukaryotes has increased its value as a model organisms for double strand break repair. We will review the S. pombe literature on double strand break repair, mainly focussing on homologous recombination in mitotic cells

The Role of the MRN Complex in the S-Phase DNA Damage Checkpoint: A Dissertation

The main focus of my work has been the role of the MRN in the S-phase DNA damage checkpoint. The MRN plays many roles in cellular metabolism; some are checkpoint dependent and some are checkpoint independent. The multiple roles in cellular metabolism complicate study of the role of the MRN in the checkpoint. MRN mutations in budding yeast and mammals may display separation of function. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5’ resection to create single stranded DNA that is required for both signaling and homologous recombination. However, it is unclear if resection is essential for all of the cellular functions of MRN. Therefore I have made mutations to mimic those in budding yeast and mammals. I found that several alleles of rad32, as well as ctp1Δ, are defective in double-strand break repair and most other functions of the complex but maintain an intact S-phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads me to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function. One of the potential roles of Rad32 and the rest of the MRN complex is in sister chromatid exchange. The genetic requirements of sister chromatid exchange have been examined using unequal sister chromatid assays which only are able to assay exchanges that are illegitimate and produce changes in the genome. Most sister chromatid exchange must be equal to maintain genomic integrity and thus far there is no good assay for equal sister chromatid exchange. Yeast cells expressing the human equilibrative nucleoside transporter 1 (hENT1) and the herpes simplex virus thymidine kinase (tk) are able to incorporate exogenous thymidine into their DNA. This strain makes it possible for the fission yeast DNA to be labeled with halogenated thymidine analogs. This strain is being used to design an assay that will label one sister with BrdU and then DNA combing will be used to see equal sister chromatid exchange

Measuring chromosome-end fusions in fission yeast

Dissertation presented to obtain the Ph.D degree in Molecular BiologyThe ends of eukaryotic chromosomes are protected from illegitimate repair by structures called telomeres. These are comprised of specific DNA repeats bound by a specialized protein complex. When telomere function is compromised, chromosome ends fuse, generating chromosomal abnormalities and genomic instability.(...

Genetic Requirements for Intra-Chromosomal Deletions

Chromosomal deletions are one of the most dangerous types of DNA damage and often arise as a result of inappropriately repaired DNA double strand breaks (DSB). These breaks are usually formed either in an induced manner from exogenous damage such as radiation, or more commonly caused from spontaneous replication errors. If there is a single strand break during replication and it is not repaired properly, as the replication fork progresses it can lead to the formation of a DSB. When there is a DSB present, there is the opportunity for a chromosomal deletion to occur. If the break is in between non-tandem direct repeats, the DNA repair machinery will degrade what is between the direct repeats through a process called Single Strand Annealing (SSA). This massive loss of DNA is what is known as a chromosomal deletion. Using an assay that in Schizosaccharomyces pombe, we can detect DSBs and determine DNA repair pathways through a selection screen of yeast cells with inactivated DNA repair genes. We generated an in vivo assay that reports exclusively SSA. We validated the assay by showing its dependence on rad52+ and independence rad51+. However, we show that earlier events epistatic to rad52+ and rad51+ have differential requirements for deletions vs. other forms of repair. Here, we delineate a more detailed epistatic pathway for intrachromosomal deletions.No embargoAcademic Major: Biolog

Role of the fission yeast Taz1p in promoting genomic stability

Telomeres define the ends of eukaryotic chromosomes and comprise multiprotein complexes bound to terminal DNA sequence repeats. An intrinsic role of telomeres is to protect chromosomal termini from being processed as damaged-induced DNA breaks; this role is critical for maintaining genomic integrity. The fission yeast Taz1 protein regulates telomere functions throughout the mitotic and meiotic cell cycles. In this study, we employed biochemical, molecular and genetic analysis to dissect the regulation and role that taz1+ and other telomere proteins play in promoting genomic stability. We find during growth at low temperatures, loss of taz1+ results in decreased viability, chromosome missegregation and DNA damage checkpoint activation. Strikingly, these cells exhibit entangled chromosomes and a pronounced de novo accumulation of DNA double strand breaks (DSBs). These defects are suppressed by altered topoisomerase II function, implicating unprotected telomeres as substrates for Top2p. Furthermore, taz1δ cells are sensitive to treatments that induce DSBs suggesting a role for Taz1p in general DSB repair. Recent data obtained from 2-D DNA gel electrophoresis suggests that Taz1 is specifically required for telomere replication and that loss of Taz1 results in perturbed fork progression through both telomere and telomere associated sequences. These data suggest a model whereby aberrant replication in taz1δ cells results in entangled chromosomes, leading to a requirement for altered Top2 activity and homologous recombination. These phenotypes are specific to taz1δ cells and not other telomere mutants suggesting that Taz1 plays a central role in promoting genomic stability that is separable from its interacting partners

What comparative genomics tells us about the evolution of the eukaryotic recombination machinery

The growing number of completely deciphered genomic sequences provides an enormous reservoir of data, which can be used for addressing questions related to functional and evolutionary biology. The wealth of this approach is documented by the fast growing numbers of recent publications in the field of evolutionary biology based on comparative genomics. Many proteins of the recombination machinery are conserved between plants, fungi and animals but some of them also show remarkable differences regarding their presence, copy number or molecular structure. For example, the protein responsible for double strand break (DSB) induction during meiosis, SPO11, which is related to the subunit A of the archaebacterial topoisomerase VI, is coded by a single gene in animals and fungi. In contrast, plants harbour three distantly related homologues, which seem to have non-redundant functions either in meiosis or in somatic cells and are indispensable for viability. Moreover, plants possess a homologue of the subunit B of the archaebacterial topoisomerase VI, not present in other eukaryotes. We also summarise the recent progress in the usage of genomic data to analyse the evolution of other DNA recombination factors. Finally, several recent studies report on a strong conservation of a reasonable number of intron positions between plants, animals and fungi. This kind of study provides a basis for comparative genomic analyses across kingdoms and demonstrates the existence of ancient introns, a topic of intensive debate

An investigation into the function of the SUMOylation of Nse2 and PCNA in S. pombe

Small ubiquitin like modifier (SUMO) is post-translationally attached to target proteins, forming a covalent bond between its C-terminal glycine and one or more lysine residues on the target protein. SUMO modification of target proteins can affect protein-protein interactions, protein activity, localistation and stability. This study set out to develop an efficient in vitro SUMOylation system to enable the identification of target lysine residues in S. pombe proteins by mass spectrometry. This involved incorporating a trypsin cleavage site adjacent to the SUMO di-glycine motif to improve peptide coverage during mass spectrometry. Several SUMOylated target proteins were identified here, including the E2 SUMO conjugating enzyme Hus5, the E3 SUMO ligase Nse2 and PCNA. The second part of this study focused on the characterisation of unSUMOylatable E3 SUMO ligase nse2 mutants. Integration of lysine to arginine mutations into the genome did not result in any mutant phenotypes and a function for auto-SUMOylation of Nse2 was not identified. During this study, human patients with mutations in the nse2 gene were reported and the equivalent mutations were integrated into the S. pombe nse2 gene to investigate the effect of the mutations. The final part of this work involved the analysis of the SUMOylation of S. pombe PCNA. Using the in vitro system, four target lysine residues for SUMO were identified. SUMOylation of PCNA was also observed in vivo following pull-down studies and 2D gel analysis of wild type and unSUMOylatable mutants. Extensive epistasis analysis was undertaken using these mutants to investigate the role of SUMOylation of S. pombe PCNA

The role of Schizosaccharomyces pombe SUMO ligases in genome stability

SUMOylation is a post-translational modification that affects a large number of proteins, many of which are nuclear. While the role of SUMOylation is beginning to be elucidated, it is clear that understanding the mechanisms that regulate the process is likely to be important. Control of the levels of SUMOylation is brought about through a balance of conjugating and deconjugating activities, i.e. of SUMO (small ubiquitin-related modifier) conjugators and ligases versus SUMO proteases. Although conjugation of SUMO to proteins can occur in the absence of a SUMO ligase, it is apparent that SUMO ligases facilitate the SUMOylation of specific subsets of proteins. Two SUMO ligases in Schizosaccharomyces pombe, Pli1 and Nse2, have been identified, both of which have roles in genome stability. We report here on a comparison between the properties of the two proteins and discuss potential roles for the proteins

Checkpoint Regulation of Replication Forks in Response to DNA Damage: A Dissertation

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

Analyses biochimiques de la recombinaison homologue méiotique chez schizosaccharomyces pombe

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010Les cassures double-brin de l'acide désoxyribonucléique (ADN) sont parmi les lésions les plus cytotoxiques car une seule lésion non réparée est létale chez la levure. Dans le but de réparer efficacement ces dommages, la cellule dispose de différents mécanismes tels que le Non Homologous End Joining (NHEJ). Toutefois ces mécanismes peuvent causer des mutations et, lorsqu' il est possible, la cellule privilégie un système qui permet une réparation très fiable: la recombinaison homologue. Ce processus peut aussi être utilisé lors de la méiose pour créer de la diversité génétique. La méiose est un mécanisme complexe et une mauvaise régulation peut mener à des problèmes tels que l' aneuploïdie. La recombinaison homologue méiotique se divise en quatre étapes majeures: (i) l' initiation qui crée des cassures double-brin par un complexe muItiprotéique incluant Rec12; (ii) la résection de l'ADN ou les protéines majeures sont Rad32/Rad50/Nbsl; (iii) l'invasion d'un duplex homologue avec les protéines RadSl et Dmcl, et pour terminer (iv) la résolution des jonctions de Holliday. Lors de mes études de doctorat, je me suis concentré sur la caractérisation biochimique des principales protéines des trois premières étapes (initiation, résection et particulièrement l'invasion) de la recombinaison méiotique chez Schizosaccharomyces pombe permettant la réparation des cassures double-brin. Mes travaux avaient pour but de caractériser les protéines Dmcl et le complexe Hop2/Mndl chez S.pombe. Nous avons déterminé que Dmcl avait comme Rad 51 la capacité de former un filament hélical sur l'ADN simple brin, ansi que de catalyser des réactions d'échange de brin. D'autre part j'ai mis en évidence le rôle que joue le complexe Hop2/Mndl dans la stimulation de Dmc 1, ainsi que les différences entre les protéines de S.pombe et de la SOurIS