Home LiteratureArticle Details
PMID: 20130019 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.

Carcinogenesis ·Vol. 31 ·No. 6 ·2010-06-00 ·Pages 994-1002

Shi W, Feng Z, Zhang J, Gonzalez-Suarez I, Vanderwaal RP, Wu X, Powell SN, Roti Roti JL, Gonzalo S, Zhang J

Abstract

Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.

MeSH Terms
Blotting, Western Cell Line, Tumor Chromosome Aberrations Comet Assay DNA Damage Genomic Instability Humans Hydroxyurea/pharmacology In Situ Hybridization, Fluorescence Phosphorylation Rad51 Recombinase/metabolism Recombination, Genetic Replication Protein A/metabolism
Chemicals
Replication Protein A RAD51 protein, human Rad51 Recombinase RPA2 protein, human Hydroxyurea
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Shi Wei
Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA.
Feng Zhihui
Zhang Jiuqin
Gonzalez-Suarez Ignacio
Vanderwaal Robert P
Wu Xiaohua
Powell Simon N
Roti Roti Joseph L
Gonzalo Susana
Zhang Junran
References (65)
65 references, click to expand
  1. Recombinant replication protein A: expression, complex formation, and functional characterization.
    J Biol Chem. 1994 Apr 15;269(15):11121-32 PMID: 8157639
  2. DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage.
    Biochemistry. 2005 Jun 14;44(23):8428-37 PMID: 15938632
  3. Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage.
    Biochem J. 2005 Nov 1;391(Pt 3):473-80 PMID: 15929725
  4. Human CtIP promotes DNA end resection.
    Nature. 2007 Nov 22;450(7169):509-14 PMID: 17965729
  5. Connecting replication and recombination.
    Mol Cell. 2003 Mar;11(3):554-6 PMID: 12667440
  6. RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint.
    J Biol Chem. 2006 Dec 22;281(51):39517-33 PMID: 17035231
  7. The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction.
    J Biol Chem. 2003 Sep 12;278(37):35584-91 PMID: 12819197
  8. The MRX complex stabilizes the replisome independently of the S phase checkpoint during replication stress.
    EMBO J. 2009 Apr 22;28(8):1142-56 PMID: 19279665
  9. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay.
    Radiat Res. 1990 Apr;122(1):86-94 PMID: 2320728
  10. The 32- and 14-kilodalton subunits of replication protein A are responsible for species-specific interactions with single-stranded DNA.
    Biochemistry. 1998 Sep 8;37(36):12496-506 PMID: 9730822
  11. ATRIP associates with replication protein A-coated ssDNA through multiple interactions.
    Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):580-5 PMID: 16407120
  12. RNA interference inhibition of Mus81 reduces mitotic recombination in human cells.
    Mol Biol Cell. 2004 Feb;15(2):552-62 PMID: 14617801
  13. The role of double-strand break repair - insights from human genetics.
    Nat Rev Genet. 2006 Jan;7(1):45-54 PMID: 16369571
  14. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex.
    EMBO J. 1992 Jun;11(6):2177-87 PMID: 1318194
  15. Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.
    J Mol Biol. 1999 Jun 25;289(5):1207-18 PMID: 10373362
  16. DNA double-strand break repair: from mechanistic understanding to cancer treatment.
    DNA Repair (Amst). 2007 Jul 1;6(7):923-35 PMID: 17363343
  17. The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A.
    EMBO J. 1997 Sep 1;16(17):5198-206 PMID: 9311980
  18. Replication protein A phosphorylation and the cellular response to DNA damage.
    DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1015-24 PMID: 15279788
  19. Rad52 protein associates with replication protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation.
    J Biol Chem. 2002 Aug 30;277(35):31663-72 PMID: 12077133
  20. Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.
    Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):11975-9 PMID: 8618826
  21. Inhibition of mammalian cell DNA synthesis by ionizing radiation.
    Int J Radiat Biol Relat Stud Phys Chem Med. 1986 May;49(5):771-81 PMID: 3516900
  22. Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.
    Nucleic Acids Res. 2009 Oct;37(18):6028-41 PMID: 19671522
  23. Requirement for RecFOR-mediated recombination in priA mutant.
    Mol Microbiol. 2004 Apr;52(2):551-62 PMID: 15066040
  24. A mutant allele of MRE11 found in mismatch repair-deficient tumor cells suppresses the cellular response to DNA replication fork stress in a dominant negative manner.
    Mol Biol Cell. 2008 Apr;19(4):1693-705 PMID: 18256278
  25. Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage.
    Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1921-6 PMID: 10051570
  26. DNA double-strand break repair: all's well that ends well.
    Annu Rev Genet. 2006;40:363-83 PMID: 16895466
  27. RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage.
    Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12903-8 PMID: 18723675
  28. Fanconi anemia (cross)linked to DNA repair.
    Cell. 2005 Dec 29;123(7):1191-8 PMID: 16377561
  29. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
    Science. 2003 Jun 6;300(5625):1542-8 PMID: 12791985
  30. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.
    Mol Cell. 2006 Jan 6;21(1):15-27 PMID: 16387650
  31. Mechanisms of replication fork restart in Escherichia coli.
    Philos Trans R Soc Lond B Biol Sci. 2004 Jan 29;359(1441):71-7 PMID: 15065658
  32. Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1.
    J Biol Chem. 2004 Nov 26;279(48):49599-608 PMID: 15371427
  33. Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.
    Mol Cell Biol. 2004 Mar;24(5):1930-43 PMID: 14966274
  34. The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.
    EMBO J. 1999 Aug 16;18(16):4498-504 PMID: 10449415
  35. ATM is required for the cellular response to thymidine induced replication fork stress.
    Hum Mol Genet. 2004 Dec 1;13(23):2937-45 PMID: 15459181
  36. BRCA1 promotes induction of ssDNA by ionizing radiation.
    Cancer Res. 2006 May 15;66(10):5181-9 PMID: 16707442
  37. Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells.
    Mol Cell Biol. 2002 Aug;22(16):5869-78 PMID: 12138197
  38. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells.
    J Biol Chem. 1997 Sep 19;272(38):23896-904 PMID: 9295339
  39. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death.
    EMBO J. 1998 Jan 15;17(2):598-608 PMID: 9430650
  40. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.
    Genes Dev. 1999 Oct 15;13(20):2633-8 PMID: 10541549
  41. scid cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase.
    Mol Cell Biol. 1995 Oct;15(10):5700-6 PMID: 7565721
  42. The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks.
    EMBO J. 2006 Oct 18;25(20):4921-32 PMID: 17036055
  43. Replication fork bypass of a pyrimidine dimer blocking leading strand DNA synthesis.
    J Biol Chem. 1997 May 23;272(21):13945-54 PMID: 9153257
  44. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism.
    Annu Rev Biochem. 1997;66:61-92 PMID: 9242902
  45. RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions.
    Biochemistry. 2003 Mar 25;42(11):3255-64 PMID: 12641457
  46. A dynamic model for replication protein A (RPA) function in DNA processing pathways.
    Nucleic Acids Res. 2006;34(15):4126-37 PMID: 16935876
  47. Protein phosphatase 2A-dependent dephosphorylation of replication protein A is required for the repair of DNA breaks induced by replication stress.
    Mol Cell Biol. 2009 Nov;29(21):5696-709 PMID: 19704001
  48. Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro.
    J Biol Chem. 1997 May 9;272(19):12634-41 PMID: 9139719
  49. Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair.
    J Biol Chem. 2007 Dec 7;282(49):35910-23 PMID: 17928296
  50. Phosphorylation of replication protein A by S-phase checkpoint kinases.
    DNA Repair (Amst). 2006 Mar 7;5(3):369-80 PMID: 16412704
  51. Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin.
    DNA Repair (Amst). 2008 Apr 2;7(4):582-96 PMID: 18289945
  52. The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.
    Mol Cell Biol. 1993 Dec;13(12):7222-31 PMID: 8246944
  53. Tracing the network connecting BRCA and Fanconi anaemia proteins.
    Nat Rev Cancer. 2004 Apr;4(4):266-76 PMID: 15057286
  54. Characterization of homologous recombination induced by replication inhibition in mammalian cells.
    EMBO J. 2001 Jul 16;20(14):3861-70 PMID: 11447127
  55. Modulation of replication protein A function by its hyperphosphorylation-induced conformational change involving DNA binding domain B.
    J Biol Chem. 2005 Sep 23;280(38):32775-83 PMID: 16006651
  56. Hyperphosphorylation of replication protein A in cisplatin-resistant and -sensitive head and neck squamous cell carcinoma cell lines.
    Head Neck. 2010 May;32(5):636-45 PMID: 19787780
  57. Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands.
    J Virol. 1988 Nov;62(11):4249-58 PMID: 2845136
  58. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.
    Nat Cell Biol. 2005 Feb;7(2):195-201 PMID: 15665856
  59. MDC1 interacts with Rad51 and facilitates homologous recombination.
    Nat Struct Mol Biol. 2005 Oct;12(10):902-9 PMID: 16186822
  60. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells.
    EMBO J. 2000 Jul 3;19(13):3398-407 PMID: 10880452
  61. Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea.
    Oncogene. 2007 Aug 23;26(39):5713-21 PMID: 17369853
  62. Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks.
    Mol Cell Biol. 2005 Aug;25(16):7158-69 PMID: 16055725
  63. Replisome assembly and the direct restart of stalled replication forks.
    Nat Rev Mol Cell Biol. 2006 Dec;7(12):932-43 PMID: 17139333
  64. Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling.
    Genes Dev. 2005 May 1;19(9):1007-12 PMID: 15879550
  65. Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks.
    DNA Repair (Amst). 2007 Jul 1;6(7):1004-17 PMID: 17409028
Article Info
Journal
Carcinogenesis
Abbr.
Carcinogenesis
ISSN
1460-2180
DOI
10.1093/carcin/bgq035
Published
2010-06-00
Epub
2010-00-03
Pages
994-1002
Language
English
Region
England
NLM ID
8008055
PMCID
PMC3916738
Subset
IM
External Links
PubMed source DOI: 10.1093/carcin/bgq035 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2