title: Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair.
abstract: 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 η (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 η-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.
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and.
authors.forename: François
authors.initials: F
authors.lastname: Bélanger
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and the Programme de Biologie Moléculaire.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Angers
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and.
authors.forename: Émile
authors.initials: É
authors.lastname: Fortier
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and.
authors.forename: Ian
authors.initials: I
authors.lastname: Hammond-Martel
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and Département d'ophtalmologie, and.
authors.forename: Santiago
authors.initials: S
authors.lastname: Costantino
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and Département de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada hugo.wurtele@umontreal.ca.
authors.forename: Elliot
authors.initials: E
authors.lastname: Drobetsky
authors.affiliation: From the Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, Québec H1T 2M4, Canada and Département de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada elliot.drobetsky@umontreal.ca.
authors.forename: Hugo
authors.initials: H
authors.lastname: Wurtele
mesh_terms: D045744:Cell Line, Tumor; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D009153:Mutagens / Q000633:toxicity; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D011740:Pyrimidine Dimers / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics*
chemical_list: D009153:Mutagens; D011740:Pyrimidine Dimers; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins
keywords: DNA replication; Saccharomyces cerevisiae; cell cycle; flow cytometry; nucleotide excision repair; ultraviolet light