Loss of Dna2 nuclease activity results in decreased Exo1-mediated resection at DNA double strand breaks.

Abstract

A DNA double strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination (HR) or non-homologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of HR, is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harbouring nuclease dead dna2-1 compared to dna2{Delta} pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harbouring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater defect in resection compared to cells where DNA2 was deleted. Both the decreased level of resection and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change, however Nej1 increased as did the frequency of alt-EJ/ MMEJ repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/564088v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c8280borg.highwire.dtl.DTLVardef@1bcf245org.highwire.dtl.DTLVardef@1c59f43org.highwire.dtl.DTLVardef@15b18d4_HPS_FORMAT_FIGEXP M_FIG C_FIG