Sgs1 helicase is required for efficient PCNA monoubiquitination and translesion DNA synthesis in Saccharomyces cerevisiae.

DNA-damage tolerance (DDT) is employed by eukaryotes to deal with replication blocks on the template strand, and is divided into two parallel pathways that are activated by sequential ubiquitination of proliferating cell nuclear antigen (PCNA) at the Lys164 residue. Rad6-Rad18-mediated PCNA monoubiquitination promotes translesion DNA synthesis (TLS) and the monoubiquitinated PCNA can be further polyubiquitinated by an Mms2-Ubc13-Rad5 complex, leading to error-free lesion bypass. We previously reported that the DNA helicase Sgs1 is required for error-free lesion bypass, probably through the double-Holliday junction migration and subsequent resolution. Surprisingly, a synthetic genetic array (SGA) screen using rev1 and rev3 as baits did not reveal an anticipated synthetic effect with sgs1, indicating a possible involvement of Sgs1 in TLS. Here, we report detailed genetic analyses demonstrating that Sgs1 plays a key role in efficient TLS and that it is probably required for the signaling of DNA damage leading to PCNA monoubiquitination. These studies collectively illustrate that Sgs1 participates in both branches of DDT and possibly plays a role in pathway choice.

Non-Smc element 5 (Nse5) of the Smc5/6 complex interacts with SUMO pathway components.

The Smc5/6 complex in Saccharomyces cerevisiae contains six essential non-Smc elements, Nse1-6. With the exception of Nse2 (also known as Mms21), which is an E3 small ubiquitin-like modifier (SUMO) ligase, very little is understood about the role of these components or their contribution to Smc5/6 functionality. Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway. Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO. SUMO also mediates the interactions between Nse5 and the two PIAS family E3 SUMO ligases, Siz1 and Siz2. Cells carrying the nse5-ts1 allele or lacking either SIZ1 or SIZ2 exhibit a reduction in Smc5 sumoylation upon MMS treatment and demonstrate functional redundancy for SUMO mediated events in the presence of DNA damage. Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.

The Mre11-Rad50-Xrs2 complex is required for yeast DNA postreplication repair.

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.

Detection of Protein Posttranslational Modifications from Whole-Cell Extracts in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an ideal model organism as numerous cellular mechanisms are conserved in mammalian cells. This includes posttranslational modifications (PTMs) such as ubiquitination, sumoylation, and phosphorylation. For ubiquitination, target proteins are readily modified via a cascade reaction, which can result in various forms of ubiquitination known to be associated with numerous cellular mechanisms. Therefore it becomes imperative for researchers to detect PTMs of their favorite proteins in order to determine how the target proteins function and are regulated. However, detection of ubiquitination in vivo, as well as some other PTMs, has proven challenging for researchers due to the presence of deconjugating enzymes in the cell. This chapter describes a step-by-step protocol on how to preserve and subsequently detect PTMs of your favorite protein from budding yeast S. cerevisiae whole-cell extracts.

The Rad5 helicase activity is dispensable for error-free DNA post-replication repair.

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and is subdivided into two parallel pathways: error-prone translesion DNA synthesis and error-free PRR. While both pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes noncanonical K63-linked polyubiquitinated PCNA to signal lesion bypass through template switch, a process thought to be dependent on Mms2-Ubc13 and a RING finger motif of the Rad5 ubiquitin ligase. Previous in vitro studies demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase activity. To investigate the genetic and mechanistic relationship between fork regression in vitro and template switch in vivo, we created and characterized site-specific mutations defective in the Rad5 RING or helicase activity. Our results indicate that both the Rad5 ubiquitin ligase and the helicase activities are exclusively involved in the same error-free PRR pathway. Surprisingly, the Rad5 helicase mutation abolishes its physical interaction with Ubc13 and the K63-linked PCNA polyubiquitin chain assembly. Indeed, physical fusions of Rad5 with Ubc13 bypass the requirement for either the helicase or the RING finger domain. Since the helicase domain overlaps with the SWI/SNF chromatin-remodelling domain, our findings suggest a structural role of this domain and that the Rad5 helicase activity is dispensable for error-free lesion bypass.

During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks.

The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.

The yeast Shu complex couples error-free post-replication repair to homologous recombination.

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis 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 screen for novel genes involved in error-free PRR and observed evidence of genetic interactions between error-free PRR and HR. Furthermore, 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 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. This mechanism appears to be conserved throughout eukaryotes.

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork.

POL32 encodes a non-essential subunit of Poldelta and plays a role in Poldelta processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polzeta-mediated translesion synthesis, but not for Poleta-mediated activity. Unlike Polzeta, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Poldelta encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1-Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.

DNA damage checkpoints are involved in postreplication repair.

Saccharomyces cerevisiae MMS2 encodes a ubiquitin-conjugating enzyme variant, belongs to the error-free branch of the RAD6 postreplication repair (PRR) pathway, and is parallel to the REV3-mediated mutagenesis branch. A mutation in genes of either the MMS2 or the REV3 branch does not result in extreme sensitivity to DNA-damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. Nevertheless, the double mutant is not as sensitive to DNA-damaging agents as a rad6 or rad18 mutant defective in the entire PRR pathway, suggesting the presence of an additional subpathway within PRR. A synthetic lethal screen was employed in the presence of a sublethal dose of a DNA-damaging agent to identify novel genes involved in PRR, which resulted in the isolation of RAD9 as a candidate PRR gene. Epistatic analysis showed that rad9 is synergistic to both mms2 and rev3 with respect to killing by methyl methanesulfonate (MMS), and the triple mutant is nearly as sensitive as the rad18 single mutant. In addition, rad9 rad18 is no more sensitive to MMS than the rad18 single mutant, suggesting that rad9 plays a role within the PRR pathway. Moreover, deletion of RAD9 reduces damage-induced mutagenesis and the mms2 spontaneous and induced mutagenesis is partially dependent on the RAD9 gene. We further demonstrated that the observed synergistic interactions apply to any two members between different branches of PRR and G1/S and G2/M checkpoint genes. These results suggest that a damage checkpoint is essential for tolerance mediated by both the error-free and error-prone branches of PRR.

Molecular basis of ataxia telangiectasia and related diseases.

Ataxia telangiectasia (AT) is a rare human disease characterized by extreme cellular sensitivity to radiation and a predisposition to cancer, with a hallmark of onset in early childhood. Several human diseases also share similar symptoms with AT albeit with different degrees of severity and different associated disorders. While all AT patients contain mutations in the AT-mutated gene (ATM), most other AT-like disorders are defective in genes encoding an MRN protein complex consisting of Mre11, Rad50 and Nbs1. Both ATM and MRN function as cellular sensors to DNA double-strand breaks, which lead to the recruitment and phosphorylation of an array of substrate proteins involved in DNA repair, apoptosis and cell-cycle checkpoints, as well as gene regulation, translation initiation and telomere maintenance. ATM is a member of the family of phosphatidylinositol 3-kinase-like protein kinases (PIKK), and the discovery of many ATM substrates provides the underlying mechanisms of heterologous symptoms among AT patients. This review article focuses on recent findings related to the initial recognition of double-strand breaks by ATM and MRN, as well as a DNA-dependent protein kinase complex consisting of the heterodimer Ku70/Ku80 and its catalytic subunit DNA-PKcs, another member of PIKK. This possible interaction suggests that a much greater complex is involved in sensing, transducing and co-ordinating cellular events in response to genome instability.