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Gene Review

RPA2  -  replication protein A2, 32kDa

Homo sapiens

Synonyms: REPA2, RF-A protein 2, RP-A p32, RP-A p34, RPA32, ...
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Disease relevance of RPA2


Psychiatry related information on RPA2

  • Facial dysmorphism, clinodactyly of the fifth finger, mental retardation and heart defects are, however, most frequently described, with a high-arched palate, in particular, seen in deletions distal to p34 [5].

High impact information on RPA2

  • Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell [6].
  • In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage) [6].
  • RPA may also have a role in modulating gene expression [6].
  • Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair [7].
  • We suggest that transcription factors may interact with RPA either to stabilize single-stranded DNA at a replication origin or to recruit DNA polymerase alpha to the replication initiation complex [1].

Chemical compound and disease context of RPA2

  • A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography [8].
  • Amino acid residues in the NH2-terminal region of the p32 protein exhibit similarity to glycoprotein X (gX) of pseudorabies virus (PRV) and its homolog in equine herpesvirus type 1 (EHV-1) [9].

Biological context of RPA2


Anatomical context of RPA2


Associations of RPA2 with chemical compounds

  • Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA-PK-dependent in CPT-treated cells and DNA-PK-independent in HU-treated cells [16].
  • In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex [17].
  • Phosphorylation of p34 RPA occurred on threonine and serine residues [15].
  • At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation [14].
  • Both human and yeast RPA stimulated the polymerase and (at subsaturating levels of RPA) the primase activities of human DNA polymerase alpha/primase on homopolymer DNA templates [18].

Physical interactions of RPA2

  • Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation [19].

Enzymatic interactions of RPA2

  • Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported [20].
  • We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase [21].
  • RPA32 was cleaved rapidly to a approximately 28-kDa polypeptide containing the C-terminus that was partially resistant to further digestion [22].
  • It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage [23].
  • RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase [24].

Regulatory relationships of RPA2

  • Multiple changes in the digestion pattern were observed when RPA bound single-stranded DNA: degradation of the approximately 52-kDa domain of RPA70 was inhibited while proteolysis of RPA32 was stimulated [22].
  • ATR knock-down with siRNA suppressed CPT-induced RPA2 hyperphosphorylation and focus formation [16].
  • Taken together, these results suggest that 53BP1 is involved in DNA damage-induced RPA2 hyperphosphorylation, and inhibition of 53BP1 function may sensitize cancer cells to camptothecin treatment [25].
  • However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported [26].
  • Further mutational studies indicated that, although the N-terminal 32-amino acid peptide of C/EBPepsilon isoform p32 did not greatly influence the transactivation activity compared with p30 isoform, this peptide does modulate transactivation activity [27].

Other interactions of RPA2

  • In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB [21].
  • In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM [20].
  • Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14 [28].
  • The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins [29].
  • UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation [20].

Analytical, diagnostic and therapeutic context of RPA2

  • Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family [30].
  • Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK [20].
  • A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame [11].
  • In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA [17].
  • In female patients but not in male patients, the body mass index z score decreased from 0.60 in group RPA0 to 0.51 in group RPA2 (P<.001) [31].


  1. The transactivator proteins VP16 and GAL4 bind replication factor A. He, Z., Brinton, B.T., Greenblatt, J., Hassell, J.A., Ingles, C.J. Cell (1993) [Pubmed]
  2. Presence of antibodies to different subunits of replication protein A in autoimmune sera. Garcia-Lozano, R., Gonzalez-Escribano, F., Sanchez-Roman, J., Wichmann, I., Nuñez-Roldan, A. Proc. Natl. Acad. Sci. U.S.A. (1995) [Pubmed]
  3. The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. Liu, V.F., Weaver, D.T. Mol. Cell. Biol. (1993) [Pubmed]
  4. The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro. Lee, S.H., Kim, D.K. J. Biol. Chem. (1995) [Pubmed]
  5. Deletion of chromosome 1p: a short review. Howard, P.J., Porteus, M. Clin. Genet. (1990) [Pubmed]
  6. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Wold, M.S. Annu. Rev. Biochem. (1997) [Pubmed]
  7. Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA. Mer, G., Bochkarev, A., Gupta, R., Bochkareva, E., Frappier, L., Ingles, C.J., Edwards, A.M., Chazin, W.J. Cell (2000) [Pubmed]
  8. Cloning and characterization of replication protein A p32 complementary DNA in zebrafish (Danio rerio). Lee, J.S., Lee, Y.S. Mar. Biotechnol. (2002) [Pubmed]
  9. Identification of an infectious laryngotracheitis virus gene encoding an immunogenic protein with a predicted M(r) of 32 kilodaltons. Kongsuwan, K., Johnson, M.A., Prideaux, C.T., Sheppard, M. Virus Res. (1993) [Pubmed]
  10. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. Shao, R.G., Cao, C.X., Zhang, H., Kohn, K.W., Wold, M.S., Pommier, Y. EMBO J. (1999) [Pubmed]
  11. A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A. Nagelhus, T.A., Haug, T., Singh, K.K., Keshav, K.F., Skorpen, F., Otterlei, M., Bharati, S., Lindmo, T., Benichou, S., Benarous, R., Krokan, H.E. J. Biol. Chem. (1997) [Pubmed]
  12. High-resolution genomic mapping of the three human replication protein A genes (RPA1, RPA2, and RPA3). Umbricht, C.B., Griffin, C.A., Hawkins, A.L., Grzeschik, K.H., O'Connell, P., Leach, R., Green, E.D., Kelly, T.J. Genomics (1994) [Pubmed]
  13. Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system. Nasheuer, H.P., von Winkler, D., Schneider, C., Dornreiter, I., Gilbert, I., Fanning, E. Chromosoma (1992) [Pubmed]
  14. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells. Zernik-Kobak, M., Vasunia, K., Connelly, M., Anderson, C.W., Dixon, K. J. Biol. Chem. (1997) [Pubmed]
  15. Characterization of ATM expression, localization, and associated DNA-dependent protein kinase activity. Gately, D.P., Hittle, J.C., Chan, G.K., Yen, T.J. Mol. Biol. Cell (1998) [Pubmed]
  16. Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks. Sakasai, R., Shinohe, K., Ichijima, Y., Okita, N., Shibata, A., Asahina, K., Teraoka, H. Genes Cells (2006) [Pubmed]
  17. Mass spectrometric identification of lysines involved in the interaction of human replication protein a with single-stranded DNA. Shell, S.M., Hess, S., Kvaratskhelia, M., Zou, Y. Biochemistry (2005) [Pubmed]
  18. An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication. Melendy, T., Stillman, B. J. Biol. Chem. (1993) [Pubmed]
  19. Coordinated regulation of replication protein A activities by its subunits p14 and p32. Weisshart, K., Pestryakov, P., Smith, R.W., Hartmann, H., Kremmer, E., Lavrik, O., Nasheuer, H.P. J. Biol. Chem. (2004) [Pubmed]
  20. Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase. Wang, H., Guan, J., Wang, H., Perrault, A.R., Wang, Y., Iliakis, G. Cancer Res. (2001) [Pubmed]
  21. Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair. Pan, Z.Q., Park, C.H., Amin, A.A., Hurwitz, J., Sancar, A. Proc. Natl. Acad. Sci. U.S.A. (1995) [Pubmed]
  22. Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA. Gomes, X.V., Henricksen, L.A., Wold, M.S. Biochemistry (1996) [Pubmed]
  23. Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis. Treuner, K., Okuyama, A., Knippers, R., Fackelmayer, F.O. Nucleic Acids Res. (1999) [Pubmed]
  24. DNA replication and the cell cycle. Stillman, B., Bell, S.P., Dutta, A., Marahrens, Y. Ciba Found. Symp. (1992) [Pubmed]
  25. 53BP1 is associated with replication protein A and is required for RPA2 hyperphosphorylation following DNA damage. Yoo, E., Kim, B.U., Lee, S.Y., Cho, C.H., Chung, J.H., Lee, C.H. Oncogene (2005) [Pubmed]
  26. Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21. Block, W.D., Yu, Y., Lees-Miller, S.P. Nucleic Acids Res. (2004) [Pubmed]
  27. Structural and functional studies of CCAAT/enhancer-binding protein epsilon. Tang, J.G., Koeffler, H.P. J. Biol. Chem. (2001) [Pubmed]
  28. The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain. Bochkareva, E., Frappier, L., Edwards, A.M., Bochkarev, A. J. Biol. Chem. (1998) [Pubmed]
  29. The 32-kilodalton subunit of replication protein A interacts with menin, the product of the MEN1 tumor suppressor gene. Sukhodolets, K.E., Hickman, A.B., Agarwal, S.K., Sukhodolets, M.V., Obungu, V.H., Novotny, E.A., Crabtree, J.S., Chandrasekharappa, S.C., Collins, F.S., Spiegel, A.M., Burns, A.L., Marx, S.J. Mol. Cell. Biol. (2003) [Pubmed]
  30. Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex. Keshav, K.F., Chen, C., Dutta, A. Mol. Cell. Biol. (1995) [Pubmed]
  31. Effects of regular physical activity on control of glycemia in pediatric patients with type 1 diabetes mellitus. Herbst, A., Bachran, R., Kapellen, T., Holl, R.W. Archives of pediatrics & adolescent medicine. (2006) [Pubmed]
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