Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.
Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
Zhu
Zhu
Z
Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Chung
Woo-Hyun
WH
Shim
Eun Yong
EY
Lee
Sang Eun
SE
Ira
Grzegorz
G
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005785:Gene Conversion; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; C028450:exodeoxyribonuclease I; D000251:Adenosine Triphosphatases; C076225:RAD5 protein, S cerevisiae; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D053484:RecQ Helicases