Distinct RPA functions promote eukaryotic DNA replication initiation and elongation

Abstract

Single-stranded DNA binding proteins (SSBs) are essential for DNA replication across all domains of life, but vary significantly in their structure and subunit composition. The eukaryotic SSB, Replication Protein A (RPA), serves critical functions in DNA replication, the DNA damage response, and DNA repair. We sought to determine the requirements for RPA during eukaryotic DNA replication initiation and elongation. To determine whether the ssDNA-binding activity is sufficient, we tested SSBs from different domains of life in reconstituted S. cerevisiae origin unwinding and DNA replication reactions. Interestingly, E. coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we found that only large, multimeric complexes with multiple DNA-binding domains support origin unwinding. In contrast, our studies demonstrated that eukaryotic replication fork function requires specific RPA domains for normal leading- and lagging-strand DNA synthesis. Together, these results reveal new requirements for ssDNA-binding proteins in eukaryotic replication origin unwinding and uncover RPA domains that are critical for faithful replication fork function.