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

through your institution Subscribe to this journal Receive 12 print issues and online access $209.00 per year only $17.42 per issue Learn more Buy this article * Purchase on SpringerLink *

Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional

subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL BASIS OF HOST PROTEIN HIJACKING IN HUMAN T-CELL LEUKEMIA VIRUS INTEGRATION Article

Open access 19 June 2020 HIV-1 REPLICATION COMPLEXES ACCUMULATE IN NUCLEAR SPECKLES AND INTEGRATE INTO SPECKLE-ASSOCIATED GENOMIC DOMAINS Article Open access 14 July 2020 STRUCTURE OF HIV-1

VPR IN COMPLEX WITH THE HUMAN NUCLEOTIDE EXCISION REPAIR PROTEIN HHR23A Article Open access 25 November 2021 REFERENCES * Fanning, E. & Knippers, R. Structure and function of simian

virus 40 large tumor antigen. _Annu. Rev. Biochem._ 61, 55–85 (1992). Article  CAS  PubMed  Google Scholar  * Bullock, P.A. The initiation of simian virus 40 DNA replication _in vitro_.

_Crit. Rev. Biochem. Mol. Biol._ 32, 503–568 (1997). Article  CAS  PubMed  Google Scholar  * Simmons, D.T. SV40 large T antigen functions in DNA replication and transformation. _Adv. Virus

Res._ 55, 75–134 (2000). Article  CAS  PubMed  Google Scholar  * Stenlund, A. Initiation of DNA replication: lessons from viral initiator proteins. _Nat. Rev. Mol. Cell Biol._ 4, 777–785

(2003). Article  CAS  PubMed  Google Scholar  * Stauffer, M.E. & Chazin, W.J. Structural mechanisms of DNA replication, repair, and recombination. _J. Biol. Chem._ 279, 30915–30918

(2004). Article  CAS  PubMed  Google Scholar  * Wold, M.S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. _Annu. Rev.

Biochem._ 66, 61–92 (1997). Article  CAS  PubMed  Google Scholar  * Iftode, C., Daniely, Y. & Borowiec, J.A. Replication protein A (RPA): the eukaryotic SSB. _Crit. Rev. Biochem. Mol.

Biol._ 34, 141–180 (1999). Article  CAS  PubMed  Google Scholar  * Bochkarev, A. & Bochkareva, E. From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. _Curr. Opin.

Struct. Biol._ 14, 36–42 (2004). Article  CAS  PubMed  Google Scholar  * Mer, G., Bochkarev, A., Chazin, W.J. & Edwards, A.M. Three-dimensional structure and function of replication

protein A. _Cold Spring Harb. Symp. Quant. Biol._ 65, 193–200 (2000). Article  CAS  PubMed  Google Scholar  * Weisshart, K. et al. Partial proteolysis of SV40 T antigen reveals

intramolecular contacts between domains and conformation changes upon hexamer assembly. _J. Biol. Chem._ 279, 38943–38951 (2004). Article  CAS  PubMed  Google Scholar  * Gai, D. et al.

Insights into the oligomeric states, conformational changes and helicase activities of SV40 large tumor antigen. _J. Biol. Chem._ 279, 38952–38959 (2004). Article  CAS  PubMed  Google

Scholar  * Dornreiter, I. et al. Interaction of DNA polymerase α-primase with cellular replication protein A and SV40 T antigen. _EMBO J._ 11, 769–776 (1992). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Melendy, T. & Stillman, B. An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA

replication. _J. Biol. Chem._ 268, 3389–3395 (1993). CAS  PubMed  Google Scholar  * Collins, K.L. & Kelly, T.J. Effects of T antigen and replication protein A on the initiation of DNA

synthesis by DNA polymerase α-primase. _Mol. Cell. Biol._ 11, 2108–2115 (1991). Article  CAS  PubMed  PubMed Central  Google Scholar  * Weisshart, K., Taneja, P. & Fanning, E. The

replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication. _J. Virol._ 72, 9771–9781 (1998). CAS  PubMed  PubMed

Central  Google Scholar  * Braun, K.A., Lao, Y., He, Z., Ingles, C.J. & Wold, M.S. Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the

activity of DNA polymerase α by multiple mechanisms. _Biochemistry_ 36, 8443–8454 (1997). Article  CAS  PubMed  Google Scholar  * Mer, G. et al. Structural basis for the recognition of DNA

repair proteins UNG2, XPA, and RAD52 by replication factor A. _Cell_ 103, 449–456 (2000). Article  CAS  PubMed  Google Scholar  * Lee, S.H. & Kim, D.K. The role of the 34-kDa subunit of

human replication protein A in simian virus 40 DNA replication _in vitro_. _J. Biol. Chem._ 270, 12801–12807 (1995). Article  CAS  PubMed  Google Scholar  * Kenny, M.K., Schlegel, U.,

Furneaux, H. & Hurwitz, J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. _J. Biol. Chem._ 265, 7693–7700 (1990)

CAS  PubMed  Google Scholar  * Matsumoto, T., Eki, T. & Hurwitz, J. Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system. _Proc. Natl. Acad.

Sci. USA_ 87, 9712–9716 (1990). Article  CAS  PubMed  PubMed Central  Google Scholar  * Arunkumar, A.I., Stauffer, M.E., Bochkareva, E., Bochkarev, A. & Chazin, W.J. Independent and

coordinated functions of replication protein A tandem high affinity single-stranded DNA binding domains. _J. Biol. Chem._ 278, 41077–41082 (2003). Article  CAS  PubMed  Google Scholar  *

Luo, X., Sanford, D.G., Bullock, P.A. & Bachovchin, W.W. Solution structure of the origin DNA–binding domain of SV40 T-antigen. _Nat. Struct. Biol._ 3, 1034–1039 (1996). Article  CAS 

PubMed  Google Scholar  * Wun-Kim, K. et al. The DNA-binding domain of simian virus 40 tumor antigen has multiple functions. _J. Virol._ 67, 7608–7611 (1993). CAS  PubMed  PubMed Central 

Google Scholar  * Simmons, D.T., Loeber, G. & Tegtmeyer, P. Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding. _J.

Virol._ 64, 1973–1983 (1990). CAS  PubMed  PubMed Central  Google Scholar  * Bradshaw, E.M. et al. T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding.

_Biochemistry_ 43, 6928–6936 (2004). Article  CAS  PubMed  Google Scholar  * Titolo, S., Welchner, E., White, P.W. & Archambault, J. Characterization of the DNA-binding properties of the

origin-binding domain of simian virus 40 large T antigen by fluorescence anisotropy. _J. Virol._ 77, 5512–5518 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yuzhakov, A.,

Kelman, Z., Hurwitz, J. & O'Donnell, M. Multiple competition reactions for RPA order the assembly of the DNA polymerase δ holoenzyme. _EMBO J._ 18, 6189–6199 (1999). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Santocanale, C., Neecke, H., Longhese, M.P., Lucchini, G. & Plevani, P. Mutations in the gene encoding the 34 kDa subunit of yeast replication

protein A cause defective S phase progression. _J. Mol. Biol._ 254, 595–607 (1995). Article  CAS  PubMed  Google Scholar  * Daughdrill, G.W. et al. Chemical shift changes provide evidence

for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A. _Nucleic Acids Res._ 31, 4176–4183 (2003). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Jackson, D., Dhar, K., Wahl, J.K., Wold, M.S. & Borgstahl, G.E. Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between

RPA32, RPA70, Rad52 and DNA. _J. Mol. Biol._ 321, 133–148 (2002). Article  CAS  PubMed  Google Scholar  * Kowalczykowski, S.C. Some assembly required. _Nat. Struct. Biol._ 7, 1087–1089

(2000). Article  CAS  PubMed  Google Scholar  * Blackwell, L.J. & Borowiec, J.A. Human replication protein A binds single-stranded DNA in two distinct complexes. _Mol. Cell. Biol._ 14,

3993–4001 (1994). Article  CAS  PubMed  PubMed Central  Google Scholar  * Blackwell, L.J., Borowiec, J.A. & Mastrangelo, I.A. Single-stranded-DNA binding alters human replication protein

A structure and facilitates interaction with DNA-dependent protein kinase. _Mol. Cell. Biol._ 16, 4798–4807 (1996). Article  CAS  PubMed  PubMed Central  Google Scholar  * Iftode, C. &

Borowiec, J.A. 5′→3′ molecular polarity of human replication protein A (RPA) binding to pseudo-origin DNA substrates. _Biochemistry_ 39, 11970–11981 (2000). Article  CAS  PubMed  Google

Scholar  * Bastin-Shanower, S.A. & Brill, S.J. Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding. _J. Biol. Chem._ 276,

36446–36453 (2001). Article  CAS  PubMed  Google Scholar  * de Laat, W.L. et al. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair. _Genes

Dev._ 12, 2598–2609 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ott, R.D., Wang, Y. & Fanning, E. Mutational analysis of simian virus 40 T-antigen primosome

activities in viral DNA replication. _J. Virol._ 76, 5121–5130 (2002). Article  CAS  PubMed  PubMed Central  Google Scholar  * Henricksen, L.A., Umbricht, C.B. & Wold, M.S. Recombinant

replication protein A: expression, complex formation, and functional characterization. _J. Biol. Chem._ 269, 11121–11132 (1994). CAS  PubMed  Google Scholar  * Pervushin, K.V., Riek, R.,

Wider, G. & Wuthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large

biological macromolecules in solution. _Proc. Natl. Acad. Sci. USA_ 94, 12366–12371 (1997). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sass, H.J., Musco, G., Stahl, S.J.,

Wingfield, P.T. & Grzesiek, S. Solution NMR of proteins within polyacrylamide gels: diffusional properties and residual alignment by mechanical stress or embedding of oriented purple

membranes. _J. Biomol. NMR_ 18, 303–309 (2000). Article  CAS  PubMed  Google Scholar  * Tycko, R., Blanco, F.J. & Ishii, Y. Alignment of biopolymers in strained gels: a new way to create

detectable dipole-dipole couplings in high-resolution biomolecular NMR. _J. Am. Chem. Soc._ 122, 9340–9349 (2000). Article  CAS  Google Scholar  * Bax, A. Weak alignment offers new NMR

opportunities to study protein structure and dynamics. _Protein Sci._ 12, 1–16 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zweckstetter, M. & Bax, A. Prediction of

sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. _J. Am. Chem. Soc._ 122, 3791–3792 (2000) Article  CAS  Google Scholar  *

Dominguez, C., Boelens, R. & Bonvin, A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. _J. Am. Chem. Soc._ 125, 1731–1737 (2003). Article

  CAS  PubMed  Google Scholar  * Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. _Acta Crystallogr. D_ 54, 905–921

(1998). Article  CAS  PubMed  Google Scholar  * Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. _J. Mol. Graph._ 14,

51–55, 29–32 (1996). Article  CAS  PubMed  Google Scholar  * Laskowski, R.A., Rullman, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking

the quality of protein structures solved by NMR. _J. Biomol. NMR_ 8, 477–486 (1996). Article  CAS  PubMed  Google Scholar  * Huang, S.G., Weisshart, K., Gilbert, I. & Fanning, E.

Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase α-primase. _Biochemistry_ 37, 15345–15352 (1998). Article  CAS

  PubMed  Google Scholar  * Simmons, D.T., Gai, D., Parsons, R., Debes, A. & Roy, R. Assembly of the replication initiation complex on SV40 origin DNA. _Nucleic Acids Res._ 32, 1103–1112

(2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. _Annu. Rev. Biochem._ 67, 721–751 (1998). Article 

CAS  PubMed  Google Scholar  Download references 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. AUTHOR INFORMATION Author notes * Alphonse I Arunkumar and Vitaly Klimovich: These authors

contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Biochemistry, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA Alphonse I Arunkumar & Walter J

Chazin * Department of Biological Sciences, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA Vitaly Klimovich, Xiaohua Jiang, Robert D Ott & Ellen Fanning * Department of

Physics, Vanderbilt University, Nashville, 37232-8725, Tennessee, USA Walter J Chazin * Department of Center for Structural Biology, Vanderbilt University, Nashville, 37232-8725, Tennessee,

USA Alphonse I Arunkumar, L Mizoue & Walter J Chazin Authors * Alphonse I Arunkumar View author publications You can also search for this author inPubMed Google Scholar * Vitaly

Klimovich View author publications You can also search for this author inPubMed Google Scholar * Xiaohua Jiang View author publications You can also search for this author inPubMed Google

Scholar * Robert D Ott View author publications You can also search for this author inPubMed Google Scholar * L Mizoue View author publications You can also search for this author inPubMed 

Google Scholar * Ellen Fanning View author publications You can also search for this author inPubMed Google Scholar * Walter J Chazin View author publications You can also search for this

author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to Ellen Fanning or Walter J Chazin. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial

interests. 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) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE 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 Download citation * Received: 02 December 2004 * Accepted: 04 March 2005 * Published: 27 March 2005 * Issue Date: 01 April 2005 * DOI:

https://doi.org/10.1038/nsmb916 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently

available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative