Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 — a key nuclease in DNA replication and repair — employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.