Essential domains of Schizosaccharomyces pombe Rad8 required for DNA damage response (original) (raw)
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Molecular Cell, 2007
Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.
2008
DNA damage may lead to mutations and loss of genome integrity. Lesions encountered during replication cause the replication machinery to stall and, unless repaired or bypased, can result in lethality of the cell. The DNA polymerase processivity clamp, PCNA (proliferating cell nuclear antigen), mediates either mutagenic damage bypass or error-fee damage avoidance through its post-translational modification states. Mono-ubiquitylated PCNA stimulates the activity of translesion DNA polymerases, while poly-ubiquitylation of PCNA is a pre-requisite for error-free damage avoidance by a yet unknown mechanism. Recent findings in the laboratory suggested that Replication Protein A (RPA), an essential single-stranded (ss) DNA-binding protein, is required for induction of PCNA ubiquitylation upon DNA damage. Consequently, the aim of my thesis was to gain further insight into the mechanism by which RPA is involved in the up-stream signals that activate PCNA modification. The Rad18 protein from ...
BMC Systems Biology, 2013
Background: The genome of living organisms is constantly exposed to several damaging agents that induce different types of DNA lesions, leading to cellular malfunctioning and onset of many diseases. To maintain genome stability, cells developed various repair and tolerance systems to counteract the effects of DNA damage. Here we focus on Post Replication Repair (PRR), the pathway involved in the bypass of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms, activated by mono-and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA). Results: We developed a novel protocol to measure the time-course ratios between mono-, di-and tri-ubiquitylated PCNA isoforms on a single western blot, which were used as the wet readout for PRR events in wild type and mutant S. cerevisiae cells exposed to acute UV radiation doses. Stochastic simulations of PCNA ubiquitylation dynamics, performed by exploiting a novel mechanistic model of PRR, well fitted the experimental data at low UV doses, but evidenced divergent behaviors at high UV doses, thus driving the design of further experiments to verify new hypothesis on the functioning of PRR. The model predicted the existence of a UV dose threshold for the proper functioning of the PRR model, and highlighted an overlapping effect of Nucleotide Excision Repair (the pathway effectively responsible to clean the genome from UV lesions) on the dynamics of PCNA ubiquitylation in different phases of the cell cycle. In addition, we showed that ubiquitin concentration can affect the rate of PCNA ubiquitylation in PRR, offering a possible explanation to the DNA damage sensitivity of yeast strains lacking deubiquitylating enzymes. Conclusions: We exploited an in vivo and in silico combinational approach to analyze for the first time in a Systems Biology context the events of PCNA ubiquitylation occurring in PRR in budding yeast cells. Our findings highlighted an intricate functional crosstalk between PRR and other events controlling genome stability, and evidenced that PRR is more complicated and still far less characterized than previously thought.
Molecular and cellular biology, 2007
The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itse...
How yeast cells deal with stalled replication forks
Current Genetics, 2020
DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.
Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae
Genes & Development, 2008
Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a ...
Post-Replication Repair Suppresses Duplication-Mediated Genome Instability
PLoS Genetics, 2010
RAD6 is known to suppress duplication-mediated gross chromosomal rearrangements (GCRs) but not single-copy sequence mediated GCRs. Here, we found that the RAD6-and RAD18-dependent post-replication repair (PRR) and the RAD5-, MMS2-, UBC13-dependent error-free PRR branch acted in concert with the replication stress checkpoint to suppress duplicationmediated GCRs formed by homologous recombination (HR). The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164. The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5. In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5. Our analysis is consistent with models in which PRR prevents replication damage from becoming double strand breaks (DSBs) and/or regulates the activity of HR on DSBs.
Journal of Molecular Biology, 2022
Genomic stability is compromised by DNA damage that obstructs replication. Rad5 plays a prominent role in DNA damage bypass processes that evolved to ensure the continuation of stalled replication. Like its human orthologs, the HLTF and SHPRH tumor suppressors, yeast Rad5 has a RING domain that supports ubiquitin ligase activity promoting PCNA polyubiquitylation and a helicase domain that in the case of HLTF and Rad5 was shown to exhibit an ATPase-linked replication fork reversal activity. The RING domain is embedded in the helicase domain, confusing their separate investigation and the understanding of the exact role of Rad5 in DNA damage bypass. Particularly, it is still debated whether the helicase domain plays a catalytic or a non-enzymatic role during error-free damage bypass and whether it facilitates a function separately from the RING domain. In this study, through in vivo and in vitro characterization of domain-specific mutants, we delineate the contributions of the two domains to Rad5 function. Yeast genetic experiments and whole-genome sequencing complemented with biochemical assays demonstrate that the ubiquitin ligase and the ATPase-linked activities of Rad5 exhibit independent catalytic activities in facilitating separate pathways during error-free lesion bypass. Our results also provide important insights into the mutagenic role of Rad5 and indicate its tripartite contribution to DNA damage tolerance.