scholarly journals Requirement of RAD52 Group Genes for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (21) ◽  
pp. 7758-7764 ◽  
Author(s):  
Venkateswarlu Gangavarapu ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Template Switching ◽  
Template Strand ◽  
Fork Regression ◽  
Strand Annealing ◽  
Almost All ◽  
Annealing Mode ◽  
Damaged Dna

ABSTRACT In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases η and ζ and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.

scholarly journals Requirement of Nse1, a Subunit of the Smc5-Smc6 Complex, for Rad52-Dependent Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (23) ◽  
pp. 8409-8418 ◽  
Author(s):  
Sergio R. Santa Maria ◽  
Venkateswarlu Gangavarapu ◽  
Robert E. Johnson ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Helicase ◽  
Template Switching ◽  
Postreplication Repair ◽  
Sumo Ligase ◽  
Fork Regression ◽  
A Subunit ◽  
Leading Strand ◽  
Dependent Pathway ◽  
Damaged Dna

ABSTRACT In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.

scholarly journals Role of Double-Stranded DNA Translocase Activity of Human HLTF in Replication of Damaged DNA

2009 ◽  
Vol 30 (3) ◽  
pp. 684-693 ◽  
Author(s):  
András Blastyák ◽  
Ildikó Hajdú ◽  
Ildikó Unk ◽  
Lajos Haracska
Keyword(s):  
Translesion Synthesis ◽  
Dna Lesions ◽  
Template Switching ◽  
Replication Forks ◽  
Double Stranded Dna ◽  
Fork Regression ◽  
Dna Translocase ◽  
Damaged Dna

ABSTRACT Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals Control of Translocations between Highly Diverged Genes by Sgs1, the Saccharomyces cerevisiae Homolog of the Bloom'sSyndrome Protein

2006 ◽  
Vol 26 (14) ◽  
pp. 5406-5420 ◽  
Author(s):  
Kristina H. Schmidt ◽  
Joann Wu ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Genome Stability ◽  
Dna Helicase ◽  
Telomere Maintenance ◽  
Template Switching ◽  
Checkpoint Kinase ◽  
Genes Encoding ◽  
Atm Protein ◽  
Break Induced Replication

ABSTRACT Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3. The S-phase checkpoint kinase and telomere maintenance factor Tel1, a homolog of the human ataxia telangiectasia (ATM) protein, prevents these translocations, whereas the checkpoint kinase Mec1, a homolog of the human ATM-related protein, and the Rad53 checkpoint kinase are not required. The translocation structures observed suggest involvement of a dicentric intermediate and break-induced replication with multiple cycles of DNA template switching.

scholarly journals Phosphorylation of Slx4 by Mec1 and Tel1 Regulates the Single-Strand Annealing Mode of DNA Repair in Budding Yeast

2007 ◽  
Vol 27 (18) ◽  
pp. 6433-6445 ◽  
Author(s):  
Sonja Flott ◽  
Constance Alabert ◽  
Geraldine W. Toh ◽  
Rachel Toth ◽  
Neal Sugawara ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Synthesis ◽  
Cell Viability ◽  
Budding Yeast ◽  
Dna Lesions ◽  
Single Strand ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Annealing Mode

ABSTRACT Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner, with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a subset of these are regulated by Mec1/Tel1-dependent phosphorylation.

scholarly journals Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Venkateswarlu Gangavarapu ◽  
Sergio R. Santa Maria ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Replication Fork ◽  
Dna Lesions ◽  
Yeast Cells ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Site ◽  
Lesion Bypass ◽  
Content Type ◽  
Replication Checkpoint ◽  
Template Strand

ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that postreplication repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.

scholarly journals DNA Length Dependence of the Single-Strand Annealing Pathway and the Role of Saccharomyces cerevisiae RAD59 in Double-Strand Break Repair

2000 ◽  
Vol 20 (14) ◽  
pp. 5300-5309 ◽  
Author(s):  
Neal Sugawara ◽  
Grzegorz Ira ◽  
James E. Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Sequence Length ◽  
Homologous Sequence ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Almost All

ABSTRACT A DNA double-strand break (DSB) created by the HO endonuclease inSaccharomyces cerevisiae will stimulate recombination between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion. Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of a larger repeat further from the DSB, which ensured that nearly all cells would survive the DSB if the smaller region was not used (N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12:563–575, 1992). Without competition, the efficiency with which homologous segments of 63 to 205 bp engaged in SSA was significantly increased. A sequence as small as 29 bp was used 0.2% of the time, and homology dependence was approximately linear up to 415 bp, at which size almost all cells survived. A mutant with a deletion of RAD59, a homologue of RAD52, was defective for SSA, especially when the homologous-sequence length was short; however, even with 1.17-kb substrates, SSA was reduced fourfold. DSB-induced gene conversion also showed a partial dependence on Rad59p, again being greatest when the homologous-sequence length was short. We found that Rad59p plays a role in removing nonhomologous sequences from the ends of single-stranded DNA when it invades a homologous DNA template, in a manner similar to that previously seen with srs2 mutants. Δrad59 affected DSB-induced gene conversion differently from msh3 and msh2, which are also defective in removing nonhomologous ends in both DSB-induced gene conversion and SSA. A msh3 rad59 double mutant was more severely defective in SSA than either single mutant.

scholarly journals Requirement of RAD5 and MMS2 for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (7) ◽  
pp. 2419-2426 ◽  
Author(s):  
Carlos A. Torres-Ramos ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translesion Synthesis ◽  
Repair Process ◽  
Postreplication Repair ◽  
Template Strand ◽  
Dna Strands ◽  
Ubiquitin Conjugating Enzyme ◽  
Uv Lesions ◽  
Enzyme Complexes ◽  
Damaged Dna

ABSTRACT UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Δ and mms2Δ mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase η (Polη) but not in the absence of REV3-encoded DNA Polζ. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polζ, error-free translesion synthesis by Polη, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a “copy choice” type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Polδ. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.

scholarly journals The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 817-831 ◽  
Author(s):  
R H Schiestl ◽  
S Prakash ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Gamma Ray ◽  
Dna Lesions ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Uv Mutagenesis ◽  
Uv Sensitivity ◽  
Suppressor Mutations ◽  
Induced Mutagenesis

Abstract rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

scholarly journals Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 103-118 ◽  
Author(s):  
Janet R Mullen ◽  
Vivek Kaliraman ◽  
Samer S Ibrahim ◽  
Steven J Brill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Protein Complexes ◽  
Ring Finger ◽  
Dna Helicase ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Helicases ◽  
Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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