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 nuclease-dead Dna2 and deletion of <i>RNH201</i>. 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 limit hybrid accumulation at DSBs and promotes DNA resection, a function not shared by Exo1. This study underscores the multifunctional roles of canonical repair factors in ensuring efficient homology-directed repair.