Replication protein A (RPA) is a heterotrimeric single stranded DNA (ssDNA)-binding protein with essential functions in DNA replication, DNA repair and DNA recombination. RPA binds with rapid kinetics and high-affinity to ssDNA with 5’ to 3’ molecular polarity. The DNA-binding domains (DBDs) A and B on the RPA1 subunit comprise the high-affinity ssDNA activity, which is also responsible for DNA unwinding. Phosphorylation of the RPA2 subunit by chicken cdc2 kinase facilitated DNA unwinding, suggesting that this post-translational modification might be important for modulating this RPA activity. RPA coated ssDNA has also been implicated as one of the triggers for intra-S phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. The human RPA1 and/or RPA2 subunits can be phosphorylated by purified checkpoint kinases ATR and ATM, the related DNA-PK, and Chk1. RPA2 is also a substrate for cyclin B-Cdc2 and cyclin A-Cdk1. Chk1 phosphorylated RPA1 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation. Further, ssDNA stimulates RPA2 phosphorylation by ATR that leads to a disassembly of the RPA heterotrimer. Phosphorylation of RPA by cyclin-Cdk has been shown to be essential for proper DNA repair in interphase cells.