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.
Highlights
Human cells are under constant assault from endogenous and environmental agents that damage DNA, leading to mutagenesis, carcinogenesis, or cell death (1)
We initially compared CPD removal in wild type (WT) versus rad4⌬ strains synchronized in G2/M using nocodazole. rad4⌬ cells are completely defective in Nucleotide excision repair (NER)-mediated CPD removal and exquisitely sensitive to UV irradiation (26)
We considered the possibility that auxin-induced degron sequence (AID)-mediated RPA depletion could alter the firing of DNA replication origins or the processivity of replication forks, thereby modulating levels of replicative stress experienced by Rfa1-AID-6FLAG-expressexpressing cells of the indicated genotype were grown to the exponential phase, fixed with formaldehyde, and examined by fluorescence microscopy
Summary
Yeast Strains and Growth Conditions—Unless stated otherwise, deletion mutants were obtained from the BY4741 haploid MATa Yeast Knock-out Collection (Thermo Scientific, YSC1053). Yeast strains were generated and propagated using standard yeast genetics methods. Mammalian Cell Culture—Culture media and supplements used in this study were purchased from Invitrogen. The SV40-transformed XPV-skin fibroblast strain XP30ROsv and its derivative expressing wild-type pol (XP30ROsv-pol/cl6), were a generous gift of Dr A. These cells were propagated in Eagle’s minimal essential medium containing 15% FBS, essential and nonessential amino acids, vitamins, L-glutamine, and antibiotics. HeLa and U2OS cells were propagated in DMEM containing 10% FBS, L-glutamine, and antibiotics. Yeast cells were washed with water and irradiated in a thin layer of water at a cell density of 0.5 OD. Monolayers were washed with PBS and irradiated through a thin layer of PBS. Immunoblotting—Whole-cell lysates of S. cerevisiae were prepared for SDS-polyacrylamide gel electrophoresis by alkaline lysis (22) or standard glass bead/trichloroacetic acid precipitation.
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