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 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 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 Stimulation of Replication Template-Switching by DNA-Protein Crosslinks

2018 ◽  
Author(s):  
Laura T. Laranjo ◽  
Julie A. Klaric ◽  
Susan T. Lovett
Keyword(s):  
Topoisomerase Ii ◽  
Template Switching ◽  
Replicative Helicase ◽  
Fork Regression ◽  
Leading Strand ◽  
Template Switch ◽  
Protein Crosslinks ◽  
Lagging Strand ◽  
Covalent Complex ◽  
Genetic Rearrangements

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-azacytidine (5azaC) is a potent mutagen for template-switching, dependent on DNA cytosine methylase (Dcm), implicating the trapped Dcm-DNA covalent complex as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require DSB repair via RecABCD. The ability to induce the SOS response is anti-mutagenic by an unknown mechanism. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.

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 Stimulation of Replication Template-Switching by DNA-Protein Crosslinks

Genes ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 14 ◽  
Author(s):  
Laura T. Laranjo ◽  
Julie A. Klaric ◽  
Leah R. Pearlman ◽  
Susan T. Lovett
Keyword(s):  
Topoisomerase Ii ◽  
Strand Break ◽  
Template Switching ◽  
Fork Regression ◽  
Leading Strand ◽  
Template Switch ◽  
Protein Crosslinks ◽  
Lagging Strand ◽  
Covalent Complex ◽  
Genetic Rearrangements

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-Azacytidine (5-azaC) is a potent mutagen for template-switching. This effect is dependent on DNA cytosine methylase (Dcm), implicating the Dcm-DNA covalent complex trapped by 5-azaC as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, which can be explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require double strand break repair via RecABCD; the ability to induce the SOS response is anti-mutagenic. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching, and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching with similar genetic properties. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.

scholarly journals The Human F-Box DNA Helicase FBH1 Faces Saccharomyces cerevisiae Srs2 and Postreplication Repair Pathway Roles

2007 ◽  
Vol 27 (21) ◽  
pp. 7439-7450 ◽  
Author(s):  
Irene Chiolo ◽  
Marco Saponaro ◽  
Anastasia Baryshnikova ◽  
Jeong-Hoon Kim ◽  
Yeon-Soo Seo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Self Regulation ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Helicase Domain ◽  
Postreplication Repair ◽  
Scf Ubiquitin Ligase ◽  
Human Orthologue ◽  
Eukaryotic Organisms

ABSTRACTTheSaccharomyces cerevisiaeSrs2 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 ofS. cerevisiae srs2mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence ofSRS2also 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 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions.

scholarly journals Yeast Rad5 Protein Required for Postreplication Repair Has a DNA Helicase Activity Specific for Replication Fork Regression

2007 ◽  
Vol 28 (1) ◽  
pp. 167-175 ◽  
Author(s):  
András Blastyák ◽  
Lajos Pintér ◽  
Ildiko Unk ◽  
Louise Prakash ◽  
Satya Prakash ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Helicase ◽  
Helicase Activity ◽  
Postreplication Repair ◽  
Fork Regression

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 Suppressor Analysis of Mutations in the 5′-Untranslated Region of COB mRNA Identifies Components of General Pathways for Mitochondrial mRNA Processing and Decay in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1315-1325
Author(s):  
Wei Chen ◽  
Maria A Islas-Osuna ◽  
Carol L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Untranslated Region ◽  
Mitochondrial Rna ◽  
Rna Turnover ◽  
Single Nucleotide ◽  
Mrna Stabilization ◽  
Recessive Mutations ◽  
A Subunit ◽  
Suppressor Analysis ◽  
Dominant Suppressor

Abstract The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5′-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5′ end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3′ → 5′ exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

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