Granger, C. | Search bioRxiv

bioRxiv2024

Dna2 nuclease resolves RNA:DNA hybrids at double-strand breaks

DNA double-strand breaks (DSBs) are highly detrimental to cells, as improper repair can result in inheritable genetic rearrangements or cell death. The role of RNA:DNA hybrids (RDHs) in DSB repair remains poorly understood, but their transient accumulation and subsequent resolution are crucial for accurate repair. The absence of the end-joining factor Nej1 at DSBs significantly reduced RDH levels, which was linked to increased activity of the Dna2 nuclease. Dna2 limits the accumulation of hybrids at DSBs, with levels rising in the presence of a nuclease-dead Dna2 and RNH201 deletion. Dna2 has a heightened preference for resolving RDHs with 5 RNA overhangs compared to duplex substrates with 5 DNA overhangs. This selective resolution by Dna2 helps restrict hybrid accumulation at DSBs and promotes resection, a function not shared by Exo1. This study underscores the multifunctional roles of canonical repair factors in ensuring efficient homology-directed repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/609401v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1578f07org.highwire.dtl.DTLVardef@16f3614org.highwire.dtl.DTLVardef@4789f1org.highwire.dtl.DTLVardef@b15f9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

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

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

biochemistry↗