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

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.

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

“…This site is also contacted by several viral replication initiator/helicase proteins, such as the simian virus 40 (SV40) T antigen (Tag) and the papillomavirus E1 (Han et al 1999;Loo and Melendy 2004;Fanning et al 2006). Studies with Tag, E1, and Pol a-primase have shown that these proteins can bind to apo-RPA or the 8-nt-mode RPA-ssDNA complex but not to the 30-nt RPA-ssDNA complex (Loo and Melendy 2004;Jiang et al 2006). It has thus been suggested that RPA-protein interactions at this site are modulated allosterically by changes in the RPA conformation or quaternary structure that are induced by the transition from the 8-to the 30-ntmode complex (Jiang et al 2006).…”

“…Studies with Tag, E1, and Pol a-primase have shown that these proteins can bind to apo-RPA or the 8-nt-mode RPA-ssDNA complex but not to the 30-nt RPA-ssDNA complex (Loo and Melendy 2004;Jiang et al 2006). It has thus been suggested that RPA-protein interactions at this site are modulated allosterically by changes in the RPA conformation or quaternary structure that are induced by the transition from the 8-to the 30-ntmode complex (Jiang et al 2006). This allosteric link is thought to be important for the RPA-mediated handoff of ssDNA to successive factors, such as the polymerase switch on Okazaki fragments (Yuzhakov et al 1999;Fanning et al 2006).…”

“…This allosteric link is thought to be important for the RPA-mediated handoff of ssDNA to successive factors, such as the polymerase switch on Okazaki fragments (Yuzhakov et al 1999;Fanning et al 2006). In addition, it is involved in the loading of RPA to ssDNA, at least in viral replication model systems (Loo and Melendy 2004;Jiang et al 2006), and may play a role in its displacement during DNA processing ).…”

Replication Protein A Stimulates the Werner Syndrome Protein Branch Migration Activity

“…3). Indeed, SV40 T antigen has been shown to actively load RPA on the unwound ssDNA strand as it is revealed (65). However, although the RPA and POT1 blockage of WRN exonuclease progression is interesting, it is not required for RPA stimulation of WRN BM activity (Fig.…”

Initiation of DNA Replication from Closed Circular DNA

“…RPA stimulates the DNA unwinding activity of T antigen (Iftode and Borowiec 1997) and an interaction with T antigen appears to be necessary for DNA replication (Weisshart, Taneja et al 1998); (Melendy and Stillman 1993). The binding of RPA70 to the T antigen DNA binding domain stimulates the association of RPA with the emerging single stranded DNA during DNA replication (Jiang, Klimovich et al 2006). Given the known sites of interaction between T antigen and RPA, it is possible to construct several models that fit the data.…”