Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex

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Abstract
We report that during activation of the simian virus 40 (SV40) pre‐replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single‐stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA‐binding domain (OBD) with the RPA high‐affinity ssDNA‐binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag‐OBD binds to RPA70AB at a site distal from the ssDNA‐binding sites and that RPA70AB, Tag‐OBD, and an 8‐nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8‐mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.