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PMID: 17110927 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

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

The EMBO journal ·Vol. 25 ·No. 23 ·2006-11-29 ·Pages 5516-26

Jiang X, Klimovich V, Arunkumar AI, Hysinger EB, Wang Y, Ott RD, Guler GD, Weiner B, Chazin WJ, Fanning E

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.

MeSH Terms
Antigens, Polyomavirus Transforming/chemistry Binding Sites DNA Replication DNA, Single-Stranded/chemistry Humans Magnetic Resonance Spectroscopy Protein Interaction Mapping Protein Structure, Quaternary Protein Structure, Tertiary Replication Origin Replication Protein A/chemistry Static Electricity
Chemicals
Antigens, Polyomavirus Transforming DNA, Single-Stranded RPA1 protein, human Replication Protein A
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Jiang Xiaohua
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232-8725, USA.
Klimovich Vitaly
Arunkumar Alphonse I
Hysinger Erik B
Wang Yingda
Ott Robert D
Guler Gulfem D
Weiner Brian
Chazin Walter J
Fanning Ellen
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1038/sj.emboj.7601432
Published
2006-11-29
Epub
2006-00-16
Pages
5516-26
Language
English
Region
England
NLM ID
8208664
PMCID
PMC1679769
Subset
IM
Grants
NIEHS NIH HHS · P30 ES000267 · United States
NIGMS NIH HHS · R01 GM065484 · United States
NCI NIH HHS · P30 CA68485 · United States
NIGMS NIH HHS · GM52948 · United States
NIGMS NIH HHS · GM65484 · United States
NCI NIH HHS · P30 CA068485 · United States
NIEHS NIH HHS · P50 ES00267 · United States
NIGMS NIH HHS · R01 GM052948 · United States
External Links
PubMed source DOI: 10.1038/sj.emboj.7601432 PMC Full Text View journal details
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