Article
- The EMBO Journal (2006) 25, 5516 - 5526
- doi:10.1038/sj.emboj.7601432
Published online: 16 November 2006
Subject Category:
Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex
Xiaohua Jiang1,a, Vitaly Klimovich1,a, Alphonse I Arunkumar2,3,a, Erik B Hysinger1, Yingda Wang1, Robert D Ott1, Gulfem D Guler1, Brian Weiner2,3, Walter J Chazin2,3,4 and Ellen Fanning1
- Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA
- Department of Biochemistry, Vanderbilt University, Nashville, TN, USA
- Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA
Correspondence to:
Ellen Fanning, Department of Biological Sciences, Vanderbilt University, 2325 Stevenson Ctr., 1161 21st Avenue South, Nashville, TN 37232-8725, USA. Tel.: +1 615 343 5677; Fax: +1 615 343 6707; E-mail: ellen.fanning@vanderbilt.edu
Walter J Chazin, Departments of Biochemistry and Chemistry and Center for Structural Biology, 5140 BIOSCI/MRBIII, Vanderbilt University, Nashville, TN 37232-8725, USA. E-mail: walter.chazin@vanderbilt.edu
aThese authors contributed equally to this work
Received 27 June 2006; Accepted 19 October 2006
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.
Keywords:
- DNA replication,
- helicase,
- replication protein A,
- SV40,
- T antigen
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