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Insights into hRPA32 C-terminal domain–mediated assembly of the simian virus 40 replisome

Nature Structural & Molecular Biology volume 12, pages 332–339 (2005)Cite this article

Abstract

Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA–binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.

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Figure 1: Monoclonal antibody 34A recognizes hRPA32C and inhibits SV40 replication.
Figure 2: The interaction of hRPA32C with Tag-OBD.
Figure 3: Effects of DNA binding and mutations on the interaction between hRPA32C and Tag-OBD.
Figure 4: Mutations in hRPA32C that weaken interaction with Tag are defective in initiation of SV40 DNA replication.
Figure 5: hRPA32C is needed for primosome activity, but not for primer extension.
Figure 6: Model for SV40 primosome activity on hRPA-coated ssDNA.

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Acknowledgements

We thank S. Bhattacharya, B. Dattilo, L. Douthitt, G. Hubbell, J. Jacob, M. Karra, M. Kenny, V. Klymovych, S. Meyn, C.S. Newlon, C. Sanders, L. Schwertman, E.M. Warren, D.R. Williams and M.S. Wold for valuable advice and assistance. Accelrys provided a gift of NMR software. Financial support is gratefully acknowledged from the US National Institutes of Health for operating grants and support to the Vanderbilt-Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology, as well as from the Howard Hughes Medical Institute Professors Program (to E.F.) and Vanderbilt University.

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Author notes

  1. Alphonse I Arunkumar and Vitaly Klimovich: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA

    Alphonse I Arunkumar & Walter J Chazin

  2. Department of Biological Sciences, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA

    Vitaly Klimovich, Xiaohua Jiang, Robert D Ott & Ellen Fanning

  3. Department of Physics, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA

    Walter J Chazin

  4. Department of Center for Structural Biology, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA

    Alphonse I Arunkumar, L Mizoue & Walter J Chazin

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Correspondence to Ellen Fanning or Walter J Chazin.

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Supplementary information

Supplementary Fig. 1

Mapping the Tag-OBD-binding site of RPA32C. (PDF 553 kb)

Supplementary Fig. 2

Mapping the RPA32C-binding site of Tag-OBD. (PDF 610 kb)

Supplementary Fig. 3

NMR analysis of side chains at the intermolecular surface. (PDF 280 kb)

Supplementary Fig. 4

NMR chemical shift analysis of the interaction of Tag-OBD with RPA32C. (PDF 287 kb)

Supplementary Fig. 5

Sequence and surface comparison of yRP32C and hRPA32C. (PDF 795 kb)

Supplementary Fig. 6

Characterization of hRPAy32C and hRPAΔ223. (PDF 81 kb)

Supplementary Fig. 7

Quantitative comparison of wild-type and mutant RPA in replication assays. (PDF 65 kb)

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Arunkumar, A., Klimovich, V., Jiang, X. et al. Insights into hRPA32 C-terminal domain–mediated assembly of the simian virus 40 replisome. Nat Struct Mol Biol 12, 332–339 (2005). https://doi.org/10.1038/nsmb916

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