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

Jiang X, Klimovich V, Arunkumar AI, Hysinger EB, Wang Y, Ott RD, Guler GD, Weiner B, Chazin WJ, Fanning E
EMBO J. 2006 25 (23): 5516-26

PMID: 17110927 · PMCID: PMC1679769 · DOI:10.1038/sj.emboj.7601432

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.

MeSH Terms (12)

Antigens, Polyomavirus Transforming Binding Sites DNA, Single-Stranded DNA Replication Humans Magnetic Resonance Spectroscopy Protein Interaction Mapping Protein Structure, Quaternary Protein Structure, Tertiary Replication Origin Replication Protein A Static Electricity

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