PDBsum entry 1z1d

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protein Protein-protein interface(s) links
Replication PDB id
Protein chains
69 a.a. *
131 a.a. *
* Residue conservation analysis
PDB id:
Name: Replication
Title: Structural model for the interaction between rpa32 c- terminal domain and sv40 t antigen origin binding domain.
Structure: Replication protein a 32 kda subunit. Chain: a. Fragment: rpa32 c-terminal domain. Synonym: rp-a, rf-a, replication factor-a protein 2, rpa32. Engineered: yes. Large t antigen. Chain: b. Fragment: sv40 t antigen origin binding domain. Synonym: sv40 t antigen.
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: rpa2, repa2, rpa32. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Simian virus 40. Organism_taxid: 10633.
NMR struc: 20 models
Authors: A.I.Arunkumar,V.Klimovich,X.Jiang,R.D.Ott,L.Mizoue, E.Fanning,W.J.Chazin
Key ref:
A.I.Arunkumar et al. (2005). Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome. Nat Struct Mol Biol, 12, 332-339. PubMed id: 15793585 DOI: 10.1038/nsmb916
03-Mar-05     Release date:   17-May-05    
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Protein chain
Pfam   ArchSchema ?
P15927  (RFA2_HUMAN) -  Replication protein A 32 kDa subunit
270 a.a.
69 a.a.
Protein chain
Pfam   ArchSchema ?
P03070  (LT_SV40) -  Large T antigen
708 a.a.
131 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     DNA replication   1 term 
  Biochemical function     DNA replication origin binding     1 term  


DOI no: 10.1038/nsmb916 Nat Struct Mol Biol 12:332-339 (2005)
PubMed id: 15793585  
Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome.
A.I.Arunkumar, V.Klimovich, X.Jiang, R.D.Ott, L.Mizoue, E.Fanning, W.J.Chazin.
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.
  Selected figure(s)  
Figure 4.
Figure 4. Mutations in hRPA32C that weaken interaction with Tag are defective in initiation of SV40 DNA replication. (a -d) Initiation of replication was tested in monopolymerase reactions containing 200 ng of the indicated hRPA and 300 or 400 (a), or 100 -400 ng (b -d), of pol-prim as indicated. Control reactions contained hRPA but lacked either pol-prim or Tag as indicated (-). The products were resolved by alkaline agarose gel electrophoresis and visualized by autoradiography. DNA size markers are indicated (M). (e) SV40 monopolymerase reactions containing radiolabeled CTP were carried out in the presence of 200 ng of the indicated hRPAs, 250 ng of pol-prim, and 250 -750 ng of Tag as indicated. Control reactions lacking Tag, hRPA or pol-prim are indicated (-). Radiolabeled RNA products were resolved by electrophoresis on a polyacrylamide gel containing 20% (w/v) urea and visualized by autoradiography. M, radiolabeled oligonucleotide size marker dT (4 -22). (f) Primer synthesis in the monopolymerase reaction was quantified and expressed as a percentage of wild-type activity. At least two reactions were used for quantification of each mutant. Brackets represent standard error.
Figure 6.
Figure 6. Model for SV40 primosome activity on hRPA-coated ssDNA. (a) hRPA (blue) is schematically depicted in the high-affinity 28 -30 nt binding mode with all four ssDNA binding domains (A -D) bound to ssDNA. hRPA14 is omitted for simplicity. The helicase domain (HEL) of a Tag hexamer (green) can associate with a pol-prim heterotetramer2, 48, 49. Antibodies against Tag that specifically inhibit either hRPA binding to Tag-OBD or pol-prim binding to the helicase domain prevent primer synthesis15. (b) We suggest that primosome assembly begins when Tag-OBD associates first with hRPA32C and then with hRPA70AB, transiently creating a short stretch of unbound ssDNA. (c) In concert with this hRPA remodeling, pol-prim associated with the Tag hexamer would be poised to access the free ssDNA and begin primer synthesis. (d) Primer extension by pol-prim is likely coupled with hRPA and Tag dissociation, and followed by the RFC- and PCNA-mediated switch to DNA polymerase 50 (not shown).
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2005, 12, 332-339) copyright 2005.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19996105 M.G.Kemp, A.C.Mason, A.Carreira, J.T.Reardon, S.J.Haring, G.E.Borgstahl, S.C.Kowalczykowski, A.Sancar, and M.S.Wold (2010).
An alternative form of replication protein a expressed in normal human tissues supports DNA repair.
  J Biol Chem, 285, 4788-4797.  
19942684 S.J.Haring, T.D.Humphreys, and M.S.Wold (2010).
A naturally occurring human RPA subunit homolog does not support DNA replication or cell-cycle progression.
  Nucleic Acids Res, 38, 846-858.  
20643958 S.Vaithiyalingam, E.M.Warren, B.F.Eichman, and W.J.Chazin (2010).
Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase.
  Proc Natl Acad Sci U S A, 107, 13684-13689.
PDB code: 3l9q
19116208 A.C.Mason, S.J.Haring, J.M.Pryor, C.A.Staloch, T.F.Gan, and M.S.Wold (2009).
An alternative form of replication protein a prevents viral replication in vitro.
  J Biol Chem, 284, 5324-5331.  
19200446 C.M.Wright, S.P.Seguin, S.W.Fewell, H.Zhang, C.Ishwad, A.Vats, C.A.Lingwood, P.Wipf, E.Fanning, J.M.Pipas, and J.L.Brodsky (2009).
Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen.
  Virus Res, 141, 71-80.  
19101707 E.Fanning, and K.Zhao (2009).
SV40 DNA replication: from the A gene to a nanomachine.
  Virology, 384, 352-359.  
19084558 G.Zeng, M.Bueno, C.J.Camachos, B.Ramaswami, C.Luo, and P.Randhawa (2009).
Validation of BKV large T-antigen ATP-binding site as a target for drug discovery.
  Antiviral Res, 81, 184-187.  
19184528 R.Janowski, S.Panjikar, A.N.Eddine, S.H.Kaufmann, and M.S.Weiss (2009).
Structural analysis reveals DNA binding properties of Rv2827c, a hypothetical protein from Mycobacterium tuberculosis.
  J Struct Funct Genomics, 10, 137-150.
PDB code: 1zel
19010961 T.R.Salas, I.Petruseva, O.Lavrik, and C.Saintomé (2009).
Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA.
  Nucleic Acids Res, 37, 38-46.  
19746968 V.Dragnea, A.I.Arunkumar, H.Yuan, D.P.Giedroc, and C.E.Bauer (2009).
Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state.
  Biochemistry, 48, 9969-9979.  
18799459 J.A.Stewart, A.S.Miller, J.L.Campbell, and R.A.Bambara (2008).
Dynamic Removal of Replication Protein A by Dna2 Facilitates Primer Cleavage during Okazaki Fragment Processing in Saccharomyces cerevisiae.
  J Biol Chem, 283, 31356-31365.  
18515800 S.K.Binz, and M.S.Wold (2008).
Regulatory functions of the N-terminal domain of the 70-kDa subunit of replication protein A (RPA).
  J Biol Chem, 283, 21559-21570.  
17287270 A.Kumar, G.Meinke, D.K.Reese, S.Moine, P.J.Phelan, A.Fradet-Turcotte, J.Archambault, A.Bohm, and P.A.Bullock (2007).
Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA.
  J Virol, 81, 4808-4818.  
17893144 B.E.Weiner, H.Huang, B.M.Dattilo, M.J.Nilges, E.Fanning, and W.J.Chazin (2007).
An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase.
  J Biol Chem, 282, 33444-33451.  
17253903 G.Meinke, P.Phelan, S.Moine, E.Bochkareva, A.Bochkarev, P.A.Bullock, and A.Bohm (2007).
The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA.
  PLoS Biol, 5, e23.
PDB codes: 2if9 2ntc
17673209 P.Taneja, I.Boche, H.Hartmann, H.P.Nasheuer, F.Grosse, E.Fanning, and K.Weisshart (2007).
Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication.
  FEBS Lett, 581, 3973-3978.  
16623697 C.J.Park, and B.S.Choi (2006).
The protein shuffle. Sequential interactions among components of the human nucleotide excision repair pathway.
  FEBS J, 273, 1600-1608.  
17005644 D.K.Reese, G.Meinke, A.Kumar, S.Moine, K.Chen, J.L.Sudmeier, W.Bachovchin, A.Bohm, and P.A.Bullock (2006).
Analyses of the interaction between the origin binding domain from simian virus 40 T antigen and single-stranded DNA provide insights into DNA unwinding and initiation of DNA replication.
  J Virol, 80, 12248-12259.  
16935876 E.Fanning, V.Klimovich, and A.R.Nager (2006).
A dynamic model for replication protein A (RPA) function in DNA processing pathways.
  Nucleic Acids Res, 34, 4126-4137.  
16611889 G.Meinke, P.A.Bullock, and A.Bohm (2006).
Crystal structure of the simian virus 40 large T-antigen origin-binding domain.
  J Virol, 80, 4304-4312.
PDB code: 2fuf
16936315 J.A.Cobb, and L.Bjergbaek (2006).
RecQ helicases: lessons from model organisms.
  Nucleic Acids Res, 34, 4106-4114.  
16216358 J.Vaynberg, and J.Qin (2006).
Weak protein-protein interactions as probed by NMR spectroscopy.
  Trends Biotechnol, 24, 22-27.  
16951253 W.Lilyestrom, M.G.Klein, R.Zhang, A.Joachimiak, and X.S.Chen (2006).
Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.
  Genes Dev, 20, 2373-2382.
PDB code: 2h1l
17110927 X.Jiang, V.Klimovich, A.I.Arunkumar, E.B.Hysinger, Y.Wang, R.D.Ott, G.D.Guler, B.Weiner, W.J.Chazin, and E.Fanning (2006).
Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.
  EMBO J, 25, 5516-5526.  
16122968 A.M.Bonvin, R.Boelens, and R.Kaptein (2005).
NMR analysis of protein interactions.
  Curr Opin Chem Biol, 9, 501-508.  
16043636 C.J.Park, J.H.Lee, and B.S.Choi (2005).
Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen.
  Nucleic Acids Res, 33, 4172-4181.
PDB code: 1ynx
16357221 J.A.Cobb, T.Schleker, V.Rojas, L.Bjergbaek, J.A.Tercero, and S.M.Gasser (2005).
Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.
  Genes Dev, 19, 3055-3069.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.