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| Subject Categories: Genome Stability & Dynamics |
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| The EMBO Journal
(2006) 25, 5516–5526, doi:10.1038/sj.emboj.7601432 Published online 16 November 2006 |
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| Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex |
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| Xiaohua Jiang1, 5, Vitaly Klimovich1, 5, Alphonse I Arunkumar2, 3, 5, Erik B Hysinger1, Yingda Wang1, Robert D Ott1, Gulfem D Guler1, Brian Weiner2, 3, Walter J Chazin2, 3, 4 and Ellen Fanning1 |
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1 Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA 2 Department of Biochemistry, Vanderbilt University, Nashville, TN, USA 3 Center for Structural Biology, Vanderbilt University, Nashville, TN, USA 4 Department of Chemistry, Vanderbilt University, Nashville, TN, USA To whom correspondence should be addressed 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 5 These authors contributed equally to this work Received 27 June 2006; Accepted 19 October 2006; Published online 16 November 2006. |
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| Abstract |
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| 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. |
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| Keywords: DNA replication, helicase, replication protein A, SV40, T antigen |
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Top of page MORE ARTICLES LIKE THISThese links to content published by NPG are automatically generated NEWS AND VIEWSNature Structural & Molecular Biology News and Views (01 Mar 2007) RESEARCH2′-O-methyl-modified RNAs Act as TLR7 Antagonists Molecular Therapy Original Article Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex The EMBO Journal Article Insights into hRPA32 C-terminal domain?mediated assembly of the simian virus 40 replisome Nature Structural & Molecular Biology Article (01 Apr 2005) |
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