Linear genomes end in characteristic structures consisting of repetitive DNA and proteins: the telomeres. These play two critical roles: on one hand they avoid the of loss of genetic information due to the incomplete replication of the chromosome ends and on the other, they provide capping structures for chromosome termini, differentiating them from double strand breaks. Telomeres contain specialized proteins (the shelterin complex), as well as proteins present elsewhere on the chromosomes (chromatin remodelling, DNA damage repair and response factors). Interestingly, several DNA damage factors are required for proper telomere maintenance, drawing a thin line between telomere protection and their recognition as broken DNA ends. Loss of telomere integrity has severe consequences for the cell, namely it can induce replicative senescence and cellular aging, or it can contribute to tumorigenesis. How telomeres are capped and how they are perceived by the cell when they become dysfunctional is essential for our understanding of the contribution of loss of telomere integrity to aging and disease. In order to unravel new factors involved in telomere maintenance, siRNA screens were performed. The optimization process has confirmed both telomeric foci and telomere dysfunction-induced foci (TIFs) as suitable readouts and the screens performed generated a list of potential candidate genes involved in telomere biology. Although some of the candidate genes tested in this work failed the validation process, other genes deserve further analysis. In addition this work also studied the role of several DNA damage factors at uncapped telomeres. Furthermore, BRCA1, CtIP and EXO1 were found to be critical for the formation of end-to-end fusions generated after TRF2 inactivation. The requirement of this proteins in this process, suggests that not only that not only the classical non-homologous end joining (C-NHEJ) pathway is active at TRF2-depelted telomeres, but emphasises the multiplicity of mechanisms that act to repair dysfunctional telomeres.

Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

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.

In Saccharomyces cerevisiae DNA postreplication repair (PRR) functions to bypass replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis (TLS) has been well characterized, little is known about the molecular events involved in error-free bypass although it has been assumed that homologous recombination (HR) is required for such a mode of lesion bypass. We undertook a genome-wide, synthetic genetic array (SGA) screen for novel genes involved in PRR and observed evidence of genetic interactions between error-free PRR and HR. We were screening for synthetic lethality which occurs when the combination of two mutations leads to an inviable organism, however, either single mutation allows for cell viability. In addition, we screened for conditionally synthetic lethal interaction which occurs when the combination of two mutations is inviable only in the presence of a DNA-damaging agent. This screen identified and assigned four genes, CSM2, PSY3, SHU1 and SHU2, whose products form a stable Shu complex, to the error-free PRR pathway. Previous studies have indicated that the Shu complex is required for efficient HR and that inactivation of any one of these genes is able to suppress the severe phenotypes of top3 and sgs1. We confirmed and further extended some of the reported observations and demonstrated that error-free PRR mutations are also epistatic to sgs1. Based on the above analyses, we propose a model in which error-free PRR utilizes the Shu complex to recruit HR to facilitate template switching, followed by double-Holliday junction resolution by Sgs1-Top3. Null mutations of HR genes including rad51, 52, 54, 55 and 57 are known to confer characteristic synergistic interactions with TLS mutations. To our surprise, null mutations of genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which is also required for HR, are epistatic to TLS mutations. The MRX complex confers an endo/exonuclease activity required for the detection and processing of DNA double-strand breaks (DSBs). Our results suggest that the MRX complex functions in both TLS and error-free PRR and that this function requires the nuclease activity of Mre11. This is in sharp contrast to other known HR genes that only function downstream of error-free PRR. Furthermore, we found that inactivation of SGS1 significantly inhibits proliferating cell nuclear antigen (PCNA) monoubiquitination and is epistatic to mutations in TLS, suggesting that Sgs1 also functions at earlier steps in DNA lesion bypass. We also examined the roles of Sae2 and Exo1, two accessory nucleases involved in DSB resection, in PRR. We found that while Sae2 is primarily required for TLS, Exo1 is exclusively involved in error-free PRR. In light of the distinct and overlapping activities of the above nucleases in the resection of DSBs, we propose that the distinct single-strand nuclease activities of MRX, Sae2 and Exo1 dictate the preference between TLS and error-free PRR for lesion bypass. While both PRR pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes non-canonical Lys63-linked polyubiquitinated PCNA to signal lesion bypass. This mechanism is dependent on the Mms2-Ubc13 complex being in close proximity to PCNA, a process thought to be dependent on Rad5. Rad5 is a member of the SWI/SNF family of ATPases that contains a RING finger motif characteristic of an E3 Ub ligase. Previous in vitro experiments demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase/ATPase activity. We therefore created site-specific mutants defective in either Rad5 RING finger or helicase/ATPase activity, or both, in order to examine their genetic interactions with known TLS and error-free PRR genes. Our results indicate that both the Rad5 RING finger motif and the helicase/ATPase activity are exclusively involved in error-free PRR. To our surprise, like the Rad5 RING finger, lack of the helicase/ATPase activity also abolishes the Lys63-linked PCNA polyubiquitin chain formation, suggesting that either the Rad5 helicase/ATPase-promoted replication fork regression signals PCNA polyubiquitination or this domain has a yet unidentified activity. In summary, results obtained from this thesis dissertation have revealed novel mechanisms of yeast PRR in S. cerevisiae, a mechanism that appears to be evolutionarily conserved throughout eukaryotes, from yeast to humans.

In Saccharomyces cerevisiae DNA postreplication repair (PRR) functions to bypass replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis (TLS) has been well characterized, little is known about the molecular events involved in error-free bypass although it has been assumed that homologous recombination (HR) is required for such a mode of lesion bypass. We undertook a genome-wide, synthetic genetic array (SGA) screen for novel genes involved in PRR and observed evidence of genetic interactions between error-free PRR and HR. We were screening for synthetic lethality which occurs when the combination of two mutations leads to an inviable organism, however, either single mutation allows for cell viability. In addition, we screened for conditionally synthetic lethal interaction which occurs when the combination of two mutations is inviable only in the presence of a DNA-damaging agent. This screen identified and assigned four genes, CSM2, PSY3, SHU1 and SHU2, whose products form a stable Shu complex, to the error-free PRR pathway. Previous studies have indicated that the Shu complex is required for efficient HR and that inactivation of any one of these genes is able to suppress the severe phenotypes of top3 and sgs1. We confirmed and further extended some of the reported observations and demonstrated that error-free PRR mutations are also epistatic to sgs1. Based on the above analyses, we propose a model in which error-free PRR utilizes the Shu complex to recruit HR to facilitate template switching, followed by double-Holliday junction resolution by Sgs1-Top3. Null mutations of HR genes including rad51, 52, 54, 55 and 57 are known to confer characteristic synergistic interactions with TLS mutations. To our surprise, null mutations of genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which is also required for HR, are epistatic to TLS mutations. The MRX complex confers an endo/exonuclease activity required for the detection and processing of DNA double-strand breaks (DSBs). Our results suggest that the MRX complex functions in both TLS and error-free PRR and that this function requires the nuclease activity of Mre11. This is in sharp contrast to other known HR genes that only function downstream of error-free PRR. Furthermore, we found that inactivation of SGS1 significantly inhibits proliferating cell nuclear antigen (PCNA) monoubiquitination and is epistatic to mutations in TLS, suggesting that Sgs1 also functions at earlier steps in DNA lesion bypass. We also examined the roles of Sae2 and Exo1, two accessory nucleases involved in DSB resection, in PRR. We found that while Sae2 is primarily required for TLS, Exo1 is exclusively involved in error-free PRR. In light of the distinct and overlapping activities of the above nucleases in the resection of DSBs, we propose that the distinct single-strand nuclease activities of MRX, Sae2 and Exo1 dictate the preference between TLS and error-free PRR for lesion bypass. While both PRR pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes non-canonical Lys63-linked polyubiquitinated PCNA to signal lesion bypass. This mechanism is dependent on the Mms2-Ubc13 complex being in close proximity to PCNA, a process thought to be dependent on Rad5. Rad5 is a member of the SWI/SNF family of ATPases that contains a RING finger motif characteristic of an E3 Ub ligase. Previous in vitro experiments demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase/ATPase activity. We therefore created site-specific mutants defective in either Rad5 RING finger or helicase/ATPase activity, or both, in order to examine their genetic interactions with known TLS and error-free PRR genes. Our results indicate that both the Rad5 RING finger motif and the helicase/ATPase activity are exclusively involved in error-free PRR. To our surprise, like the Rad5 RING finger, lack of the helicase/ATPase activity also abolishes the Lys63-linked PCNA polyubiquitin chain formation, suggesting that either the Rad5 helicase/ATPase-promoted replication fork regression signals PCNA polyubiquitination or this domain has a yet unidentified activity. In summary, results obtained from this thesis dissertation have revealed novel mechanisms of yeast PRR in S. cerevisiae, a mechanism that appears to be evolutionarily conserved throughout eukaryotes, from yeast to humans.

No description available.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.