title: RPA coordinates DNA end resection and prevents formation of DNA hairpins.
abstract: Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11Δ to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Huan
authors.initials: H
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Lisby
authors.forename: Lorraine S
authors.initials: LS
authors.lastname: Symington
mesh_terms: D015153:Blotting, Western; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; C522758:Pol32 protein, S cerevisiae; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; C028450:exodeoxyribonuclease I; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D053484:RecQ Helicases