A Role for Checkpoint Kinase-Dependent Rad26 Phosphorylation in Transcription-Coupled DNA Repair in <i>Saccharomyces cerevisiae</i>

Täschner, Michael; Harreman, Michelle T.; Teng, Yumin; Gill, Hefin; Anindya, Roy; Skehel, J. Mark; Waters, Raymond; Svejstrup, Jesper Q.

doi:10.1128/mcb.00822-09

Cited by 19 publications

(17 citation statements)

References 67 publications

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“…reflecting inhibition of both TC-NER and GG-NER, respectively) (20). This investigation did not measure repair as a function of cell cycle, although the extent of the defect as observed in cycling cells presumably indicates, in contrast with our own results, significant inhibition of NER during all phases in Mec1-deficient cells.…”

Section: Discussioncontrasting

confidence: 73%

“…Yeast strains were generated and propagated using standard yeast genetics methods. Expression plasmids for RAD26 and rad26 S27A were kindly provided by Dr. J. Q. Svejstrup (20). For cell synchronization in G 2 /M, cultures were diluted to a cell density of 0.5 OD and incubated with 15 g/ml nocodazole (Cedarlane; 1% DMSO final concentration) for 3 h at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…Deletion of SML1, encoding an inhibitor of the ribonucleotide reductase enzymatic complex, is required to permit viability of mec1⌬ cells (27). Rad26 is the yeast homologue of the human TC-NERspecific lesion recognition factor CSB and was previously shown to be phosphorylated by Mec1 on serine 27 (20). To facilitate comparison between different mutant strains that may traverse the G 1 -S transition with varying speeds, cells were synchronized in G 1 with ␣-factor and released toward S phase in the presence of 200 mM hydroxyurea (HU) for 60 min prior to UV treatment.…”

Section: Mec1 Is Required For Efficient Ner Uniquely During S Phase Imentioning

confidence: 99%

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Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Bart

Angers

Fortier

et al. 2016

Journal of Biological Chemistry

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Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1-3 overexpression rescues such deficiency in either ATR-or polymerase -deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.

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Section: Discussioncontrasting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

Section: Mec1 Is Required For Efficient Ner Uniquely During S Phase Imentioning

confidence: 99%

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Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Bart

Angers

Fortier

et al. 2016

Journal of Biological Chemistry

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“…First, a connection between DDR components and the efficiency of NER has been previously reported. In yeast, for example, there is a moderate reduction in the rate of CPD removal in rad9 (Al-Moghrabi et al 2001) and mec1 mutants, but not in rad53 or chk1 mutants (Taschner et al 2010). A significant reduction in the rate of removing CPDs and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) has been reported in ATR (the yeast Mec1 homolog)-deficient human fibroblasts (Auclair et al 2008), and a moderate reduction in the rate of CPD removal has been observed in human cells carrying the XPA (the yeast Rad14 homolog) S196A allele that lacks an ATR-phosphorylation site (Shell et al 2009).…”

Section: Discussionmentioning

confidence: 99%

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

Kozmin

Jinks-Robertson

2013

Genetics

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Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colonycolor system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol h translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol z-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. MUTAGENESIS associated with induced DNA damage is generally considered to occur during S phase, when polymerase-blocking lesions are bypassed in an error-prone manner by a translesion synthesis (TLS) DNA polymerase. In the yeast Saccharomyces cerevisiae, ultraviolet (UV)-induced mutagenesis is dependent on Pol z (Rev3-Rev7) and Rev1, with rev1, rev3, or rev7 mutants exhibiting a "reversionless" phenotype in response to UV irradiation (Lawrence 2002). Just as induced mutagenesis is considered to be a consequence of error-prone TLS, nucleotide excision repair (NER) is thought to counteract such mutagenesis by removing UV-induced lesions before they can be encountered during DNA replication.Although NER is generally considered to be an error-free, mutation-avoidance mechanism, data obtained .30 years ago demonstrated a pro-mutation role of NER in the UVinduced mutagenesis that occurs in nondividing yeast cells (Eckardt and Haynes 1977;James and Kilbey 1977;James et al. 1978;Eckardt et al. 1980). Either the induction of recessive lethal mutations in diploid strains (James and Kilbey 1977;James et al. 1978) or the induction of forward mutations in the de novo adenine biosynthetic pathway in haploid strains was monitored (Eckardt and Haynes 1977;Eckardt et al. 1980). Despite the differences in assay systems used, the authors reached the same conclusions. First, UVinduced mutations in nondi...

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“…We are currently investigating the mechanism by which the H2B deubiquitylases are activated by stalled RNA Pol II. It is interesting to note that activation of the yeast TCR protein Rad26 (an otholog to mammalian Cockayne syndrome group B protein, CSB) by UV damage requires its phosphorylation [21] . A similar mechanism may also apply to the activation of H2B deubiquitylases upon RNA Pol II stalling.…”

Section: Research Highlightmentioning

confidence: 99%

Rescue of DNA damage-stalled RNA Pol II: histone H2B in action

2015

RNA Dis

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RNA Pol II elongation in eukaryotes is coupled with a series of histone modifications. Elongating RNA Pol II can be strongly stalled by lesions on the DNA template. However, it is unclear whether RNA Pol II stalling affects elongation-associated histone modifications. We have explored this important question by investigating the function of histone H2B mono-ubiquitylation (H2Bub), a well-characterized epigenetic mark associated with RNA Pol II elongation, in the cellular response to DNA lesions induced by ultraviolet (UV) radiation. We found that, in contrast to transcription elongation, RNA Pol II stalling induced by UV lesions triggers rapid and significant H2B deubiquitylation that removes ubiquitin from H2B. Interestingly, in yeast mutant cells that lack H2B deubiquitylation enzymes, rescue of the stalled RNA Pol II by transcription-coupled repair (TCR) is significantly impaired. Thus, our study has established a direct connection between RNA Pol II stalling and a histone modification response.

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A Role for Checkpoint Kinase-Dependent Rad26 Phosphorylation in Transcription-Coupled DNA Repair in Saccharomyces cerevisiae

Cited by 19 publications

References 67 publications

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

Rescue of DNA damage-stalled RNA Pol II: histone H2B in action

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