title: Synthetic viability genomic screening defines Sae2 function in DNA repair.
abstract: DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Fabio
authors.initials: F
authors.lastname: Puddu
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Tobias
authors.initials: T
authors.lastname: Oelschlaegel
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Guerini
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Geisler
authors.affiliation: Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Hengyao
authors.initials: H
authors.lastname: Niu
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK The Wellcome Trust Sanger Institute, Hinxton Cambridge, UK.
authors.forename: Mareike
authors.initials: M
authors.lastname: Herzog
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Israel
authors.initials: I
authors.lastname: Salguero
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Bernardo
authors.initials: B
authors.lastname: Ochoa-Montaño
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Viré
authors.affiliation: Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Sung
authors.affiliation: The Wellcome Trust Sanger Institute, Hinxton Cambridge, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.affiliation: The Wellcome Trust Sanger Institute, Hinxton Cambridge, UK.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Keane
authors.affiliation: The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK The Wellcome Trust Sanger Institute, Hinxton Cambridge, UK s.jackson@gurdon.cam.ac.uk.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jackson
mesh_terms: D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D000099257:Meiotic Recombination Protein SPO11
chemical_list: D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D029701:Saccharomyces cerevisiae Proteins; C107849:SAE2 protein, S cerevisiae; C094191:MRE11 protein, S cerevisiae; C518938:Spo11 protein, S cerevisiae; D000099257:Meiotic Recombination Protein SPO11
keywords: Mre11; Sae2; suppressor screening; synthetic viability; whole‐genome sequencing