Mechanism of Dynamic Binding of Replication Protein A to ssDNA.

Replication protein A (RPA) serves as a hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with single-stranded DNA (ssDNA) intermediates before handing them over to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means that RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular dynamics simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the length of ssDNA intermediates. We further estimated the association kinetics that matches excellently well with previous experimental studies and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive "bulge" formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. This unanticipated binding mechanism successfully explains how the "short-lived", long ssDNA intermediates are processed quickly in vivo. This study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication.