title: The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1.
abstract: Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1(BLM). How Exo1 and Dna2-Sgs1(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.
authors.affiliation: Institute for Cell and Molecular Biosciences (ICaMB), Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Greg H P
authors.initials: GH
authors.lastname: Ngo
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: Lata
authors.initials: L
authors.lastname: Balakrishnan
authors.affiliation: Institute for Cell and Molecular Biosciences (ICaMB), Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Marion
authors.initials: M
authors.lastname: Dubarry
authors.affiliation: Divisions of Biology and Chemistry, Caltech, Braun Laboratories, Pasadena, CA 91125, USA.
authors.forename: Judith L
authors.initials: JL
authors.lastname: Campbell
authors.affiliation: Institute for Cell and Molecular Biosciences (ICaMB), Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK d.a.lydall@ncl.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Lydall
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; C509908:DNA2 protein, human; D053484:RecQ Helicases