Abstract
Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity (KD ≈ 100 pm). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart.
Highlights
Replication protein A (RPA) is a major ssDNA-binding protein in eukaryotes [1]
We found that pol h was the most efficient in utilizing R-loops for initiation of DNA synthesis, but most of its products were short, 83 nt, as could be expected for translesion DNA polymerases [30] (Fig. 5B, lane 3)
We identified novel unanticipated activities of RPA: high-affinity binding to RNA and formation of R-loops between RNA and homologous supercoiled dsDNA
Summary
Replication protein A (RPA) is a major ssDNA-binding protein in eukaryotes [1]. It is a highly conserved trimeric protein composed of three subunits, RPA70, RPA32, and RPA14, which all are essential for cell viability [2]. We identified novel unanticipated activities of RPA: high-affinity binding to RNA and formation of R-loops between RNA and homologous supercoiled dsDNA.
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