Regulation of hetDNA Length during Mitotic Double-Strand Break Repair in Yeast

Heteroduplex DNA (hetDNA) is a key molecular intermediate during the repair of mitotic double-strand breaks by homologous recombination, but its relationship to 5' end resection and/or 3' end extension is poorly understood. In the current study, we examined how perturbations in these processes affect the hetDNA profile associated with repair of a defined double-strand break (DSB) by the synthesis-dependent strand-annealing (SDSA) pathway. Loss of either the Exo1 or Sgs1 long-range resection pathway significantly shortened hetDNA, suggesting that these pathways normally collaborate during DSB repair. In addition, altering the processivity or proofreading activity of DNA polymerase δ shortened hetDNA length or reduced break-adjacent mismatch removal, respectively, demonstrating that this is the primary polymerase that extends both 3' ends. Data are most consistent with the extent of DNA synthesis from the invading end being the primary determinant of hetDNA length during SDSA.