Repository logo
 

Synthetic viability genomic screening defines Sae2 function in DNA repair.


Type

Article

Change log

Authors

Oelschlaegel, Tobias 
Guerini, Ilaria 
Geisler, Nicola J 
Niu, Hengyao 

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.

Description

Keywords

Mre11, Sae2, suppressor screening, synthetic viability, whole‐genome sequencing, DNA Repair, DNA, Fungal, DNA, Single-Stranded, Endodeoxyribonucleases, Endonucleases, Exodeoxyribonucleases, High-Throughput Nucleotide Sequencing, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins

Journal Title

EMBO J

Conference Name

Journal ISSN

0261-4189
1460-2075

Volume Title

34

Publisher

Wiley-VCH Verlag
Sponsorship
Wellcome Trust (101126/Z/13/Z)
Cancer Research Uk (None)
Wellcome Trust (092096/Z/10/Z)
Cancer Research Uk (None)
We thank M.P. Longhese, R. Rothstein and J. Haber for providing strains and plasmids; Sir T. Blundell and T. Ochi for advice on structural biology and for providing comments to the manuscript. Research in the Jackson laboratory is funded by Cancer Research UK Programme Grant C6/A11224, the European Research Council and the European Community Seventh Framework Programme Grant Agreement No. HEALTH‐F2‐2010‐259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). SPJ receives his salary from the University of Cambridge, UK, supplemented by CRUK. TO, IG and FP were funded by Framework Programme Grant Agreement No. HEALTH‐F2‐2010‐259893 (DDResponse). FP also received funding from EMBO (Fellowship ALTF 1287‐2011); NG and IS are funded by the Wellcome Trust (101126/Z/13/Z). DJA and TMK were supported by Cancer Research UK and the Wellcome Trust (WT098051). PS and HN were supported by NIH grants RO1ES007061 and K99ES021441, respectively.