Home LiteratureArticle Details
PMID: 17583916 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Structural characterization of human RPA sequential binding to single-stranded DNA using ssDNA as a molecular ruler.

Biochemistry ·Vol. 46 ·No. 28 ·2007-07-17 ·Pages 8226-33

Cai L, Roginskaya M, Qu Y, Yang Z, Xu Y, Zou Y

Abstract

Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA-ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Using a set of oligonucleotides (dT) with varying lengths as a molecular ruler and also as the substrate, we have determined at single-nucleotide resolution the relative positions of the ssDNA with interacting intrinsic tryptophans of RPA. Our results revealed that Trp528 in DBD-C and Trp107 in DBD-D contact ssDNA at the 16th and 24th nucleotides (nt) from the 5'-end of the substrate, respectively. Evaluation of the relative spatial arrangement of RPA domains in the RPA-ssDNA complex suggested that DBD-B and DBD-C are spaced by about 4 nt ( approximately 19 A) apart, whereas DBD-C and DBD-D are spaced by about 7 nt ( approximately 34 A). On the basis of these geometric constraints, a global structure model for the binding of the major RPA DBDs to ssDNA was proposed.

MeSH Terms
Binding Sites DNA, Single-Stranded/chemistry,metabolism Humans Models, Molecular Mutation Protein Binding Protein Structure, Tertiary Replication Protein A/chemistry,genetics,metabolism Spectrometry, Fluorescence
Chemicals
DNA, Single-Stranded RPA1 protein, human Replication Protein A RPA2 protein, human
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Cai Lifeng
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Roginskaya Marina
Qu Youxing
Yang Zhengguan
Xu Ying
Zou Yue
References (47)
47 references, click to expand
  1. 5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates.
    Biochemistry. 2000 Oct 3;39(39):11970-81 PMID: 11009611
  2. From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold.
    Curr Opin Struct Biol. 2004 Feb;14(1):36-42 PMID: 15102447
  3. Polarity of human replication protein A binding to DNA.
    Nucleic Acids Res. 2001 Jan 15;29(2):373-9 PMID: 11139606
  4. Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding.
    EMBO J. 2001 Feb 1;20(3):612-8 PMID: 11157767
  5. The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding.
    Nucleic Acids Res. 2001 Aug 1;29(15):3270-6 PMID: 11470885
  6. Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding.
    J Biol Chem. 2001 Sep 28;276(39):36446-53 PMID: 11479296
  7. Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA.
    EMBO J. 2002 Apr 2;21(7):1855-63 PMID: 11927569
  8. Dimerization of human XPA and formation of XPA2-RPA protein complex.
    Biochemistry. 2002 Oct 29;41(43):13012-20 PMID: 12390028
  9. The Xplor-NIH NMR molecular structure determination package.
    J Magn Reson. 2003 Jan;160(1):65-73 PMID: 12565051
  10. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
    Science. 2003 Jun 6;300(5625):1542-8 PMID: 12791985
  11. NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair.
    Nucleic Acids Res. 2003 Aug 15;31(16):4747-54 PMID: 12907715
  12. 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
  13. Independent and coordinated functions of replication protein A tandem high affinity single-stranded DNA binding domains.
    J Biol Chem. 2003 Oct 17;278(42):41077-82 PMID: 12881520
  14. Replication protein A-mediated recruitment and activation of Rad17 complexes.
    Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13827-32 PMID: 14605214
  15. Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.
    Mol Cell Biol. 2004 Mar;24(5):1930-43 PMID: 14966274
  16. Human replication protein A binds single-stranded DNA in two distinct complexes.
    Mol Cell Biol. 1994 Jun;14(6):3993-4001 PMID: 8196638
  17. Interactions of human replication protein A with oligonucleotides.
    Biochemistry. 1994 Nov 29;33(47):14197-206 PMID: 7947831
  18. Recombinant human replication protein A binds to polynucleotides with low cooperativity.
    Biochemistry. 1995 Feb 14;34(6):2058-64 PMID: 7849064
  19. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair.
    Nature. 1995 Apr 6;374(6522):566-9 PMID: 7700386
  20. Replication protein A induces the unwinding of long double-stranded DNA regions.
    J Mol Biol. 1996 May 31;259(1):104-12 PMID: 8648638
  21. Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase.
    Mol Cell Biol. 1996 Sep;16(9):4798-807 PMID: 8756638
  22. Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA.
    Nature. 1997 Jan 9;385(6612):176-81 PMID: 8990123
  23. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism.
    Annu Rev Biochem. 1997;66:61-92 PMID: 9242902
  24. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.
    Genes Dev. 1998 Aug 15;12(16):2598-609 PMID: 9716411
  25. Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A.
    Mol Cell Biol. 1998 Dec;18(12):7225-34 PMID: 9819409
  26. Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3'-end of nascent DNA.
    FEBS Lett. 1999 Apr 30;450(1-2):131-4 PMID: 10350071
  27. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.
    Annu Rev Biochem. 2004;73:39-85 PMID: 15189136
  28. Replication protein A phosphorylation and the cellular response to DNA damage.
    DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1015-24 PMID: 15279788
  29. Binding properties of replication protein A from human and yeast cells.
    Mol Cell Biol. 1992 Jul;12(7):3050-9 PMID: 1320195
  30. 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
  31. 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
  32. Replication protein A (RPA): the eukaryotic SSB.
    Crit Rev Biochem Mol Biol. 1999;34(3):141-80 PMID: 10473346
  33. RPA subunit arrangement near the 3'-end of the primer is modulated by the length of the template strand and cooperative protein interactions.
    Nucleic Acids Res. 1999 Nov 1;27(21):4235-40 PMID: 10518616
  34. Human replication protein A: global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker.
    J Biomol NMR. 1999 Aug;14(4):321-31 PMID: 10526407
  35. Interactions of human replication protein A with single-stranded DNA adducts.
    Biochem J. 2005 Jan 15;385(Pt 2):519-26 PMID: 15362978
  36. Mass spectrometric identification of lysines involved in the interaction of human replication protein a with single-stranded DNA.
    Biochemistry. 2005 Jan 25;44(3):971-8 PMID: 15654753
  37. Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage.
    Biochemistry. 2005 May 17;44(19):7361-8 PMID: 15882075
  38. 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
  39. DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair.
    Biochemistry. 2005 Jun 14;44(23):8438-48 PMID: 15938633
  40. Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells.
    Oncogene. 2005 Jul 7;24(29):4728-35 PMID: 15897895
  41. 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
  42. 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
  43. Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses.
    J Cell Physiol. 2006 Aug;208(2):267-73 PMID: 16523492
  44. A dynamic model for replication protein A (RPA) function in DNA processing pathways.
    Nucleic Acids Res. 2006;34(15):4126-37 PMID: 16935876
  45. 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
  46. Thermodynamic characterization of the interaction of mutant UvrB protein with damaged DNA.
    Biochemistry. 2004 Apr 13;43(14):4206-11 PMID: 15065864
  47. Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA.
    Cell. 2000 Oct 27;103(3):449-56 PMID: 11081631
Article Info
Journal
Biochemistry
Abbr.
Biochemistry
ISSN
0006-2960
DOI
10.1021/bi7004976
Published
2007-07-17
Epub
2007-00-21
Pages
8226-33
Language
English
Region
United States
NLM ID
0370623
PMCID
PMC2553558
Subset
IM
Grants
NCI NIH HHS · R01 CA086927-05 · United States
NCI NIH HHS · R01 CA086927 · United States
NCI NIH HHS · R01 CA086927-04 · United States
NCI NIH HHS · R56 CA086927 · United States
NCI NIH HHS · CA86927 · United States
NCI NIH HHS · R01 CA086927-03 · United States
External Links
PubMed source DOI: 10.1021/bi7004976 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: product@genelibs.com

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