Biochemical impact of p300-mediated acetylation of replication protein A: Implications for DNA metabolic pathway choice

Abstract

Replication Protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair, and cell cycle checkpoint activation. Binding of RPA to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation, and suppresses illegitimate recombination. In our current study, we identified the acetyltransferase p300 to be capable of acetylating the 70 kDa subunit of RPA in vitro and within cells. The acetylation status of RPA changes throughout the cell cycle, increasing during the S and G2/M phases, and after UV-induced damage. Furthermore, we were able to specifically identify RPA directly associated with the replication fork during the S phase and UV damage to be acetylated. Based on these observations, we evaluated the impact of lysine acetylation on the biochemical properties of RPA. Investigation of binding properties of RPA revealed that acetylation of RPA increased its binding affinity to ssDNA compared to unmodified RPA. The improvement in binding efficiency was a function of DNA length with the greatest increases observed on shorter length ssDNA oligomers. Enzymatic assays further revealed that upon acetylation RPA governs the switch between the short and long flap pathway for Okazaki fragment processing. Our findings demonstrate that p300-dependent, site-specific acetylation enhances RPA's DNA binding properties, potentially regulating its function during various DNA transactions.