In vitro analysis of the zinc-finger motif in human replication protein A

Dong, Jiaowang; Park, Jang Su; Lee, Suk Hee

doi:10.1042/bj3370311

Cited by 26 publications

(10 citation statements)

References 49 publications

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“…For this, wild-type RPA and two RPA mutants (RPAp34C33 lacking the C terminus of p34 subunit (XPA interaction domain); ZFM4, a mutant with cysteine to alanine substitution at the zinc finger domain of the p70 subunit) were compared in the displacement of XPC-hHR23B from damaged DNA. Although both mutants poorly supported NER activity in vitro (14,36), ZFM4 supported the displacement of XPC-hHR23B from damaged DNA, whereas RPAp34C33 did not (Fig. 7).…”

Section: Figmentioning

confidence: 94%

Biochemical Analysis of the Damage Recognition Process in Nucleotide Excision Repair

You

Wang²,

Lee

2003

Journal of Biological Chemistry

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XPA, XPC-hHR23B, RPA, and TFIIH all are the damage recognition proteins essential for the early stage of nucleotide excision repair. Nonetheless, it is not clear how these proteins work together at the damaged DNA site. To get insight into the molecular mechanism of damage recognition, we carried out a comprehensive analysis on the interaction between damage recognition proteins and their assembly on damaged DNA. XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex. Instead, XPC-hHR23B was effectively displaced from the damaged DNA by the combined action of RPA and XPA. A mutant RPA lacking the XPA interaction domain failed to displace XPChHR23B from damaged DNA, suggesting that XPA and RPA cooperate with each other to destabilize the XPChHR23B-damaged DNA complex. Interestingly, the presence of hHR23B significantly increased RPA/XPA-mediated displacement of XPC from damaged DNA, suggesting that hHR23B may modulate the binding of XPC to damaged DNA. Together, our results suggest that damage recognition occurs in a multistep process such that XPC-hHR23B initiates damage recognition, which was replaced by combined action of XPA and RPA. XPA and RPA, once forming a complex at the damage site, would likely work with TFIIH, XPG, and ERCC1-XPF for dual incision.

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Section: Figmentioning

confidence: 94%

Biochemical Analysis of the Damage Recognition Process in Nucleotide Excision Repair

You

Wang²,

Lee

2003

Journal of Biological Chemistry

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“…The occluded binding site for this domain is approximately 8 nucleotides (nt) on ssDNA (4). A third ssDNA-binding motif, DBD-C, is located within the carboxyl terminus of RPA70, along with a zinc finger structure that has been found to be important for structural stability and the ssDNA-binding activity of RPA (7,10,20,74). RPA32 also binds ssDNA via a fourth ssDNA-binding motif, DBD-D (3,6,59).…”

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confidence: 99%

Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Loo

Melendy

2004

J Virol

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With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.The Papillomavirinae are small, nonenveloped viruses with double-stranded, circular DNA genomes. With the exception of two virally encoded proteins, E1 and E2, papillomavirus (PV) DNA replication is carried out entirely by the host cellular replication machinery (reviewed in references 14, 42, and 68). A partially reconstituted cell-free system of replication has allowed the identification of many of these cellular factors (34,49,55,57). Among them are cellular factors that were previously found to be necessary and sufficient for simian virus 40 (SV40) DNA replication, including DNA polymerase alphaprimase (pol-prim) and replication protein A (RPA) (reviewed in references 13, 51, and 67). However, unlike SV40, additional cellular factors, some of which have yet to be identified, are required for efficient PV DNA replication (44,46,49). Nevertheless, many parallels have been drawn between the two viral systems, particularly regarding the initiation and elongation of DNA replication.The PV E1 protein is an ATP-dependent viral DNA helicase (77); E2 is a multifunctional regulator of viral transcription and replication (18,26,35,48). PV DNA replication is initiated by the assembly of E1 as a double hexamer at the origin of replication, resulting in a localized melting of the DNA template (21, 63). The sequence-specific binding of E1 to the origin is directed by its interactions with E2 (45,54,61)...

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“…The replication protein A (RPA 1 ; also known as human single-stranded DNA-binding protein) is a three-subunit complex (70-, 34-, and 11-kDa; p70, p34, and p11, respectively) essential for DNA replication, nucleotide excision repair, and genetic recombination (54). In simian virus 40 (SV40) replication, RPA mediates unwinding of replication origin in the presence of SV40 T-antigen and topoisomerase I/II.…”

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confidence: 99%

Zinc Finger of Replication Protein A, a Non-DNA Binding Element, Regulates Its DNA Binding Activity through Redox

Park

Wang

Park

et al. 1999

Journal of Biological Chemistry

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though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124 -15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reductionoxidation (redox) through its zinc finger domain. RPAssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.The replication protein A (RPA 1 ; also known as human single-stranded DNA-binding protein) is a three-subunit complex (70-, 34-, and 11-kDa; p70, p34, and p11, respectively) essential for DNA replication, nucleotide excision repair, and genetic recombination (54). In simian virus 40 (SV40) replication, RPA mediates unwinding of replication origin in the presence of SV40 T-antigen and topoisomerase I/II. During replication, it interacts with SV40 T-antigen and DNA polymerase ␣-primase (pol ␣-primase) complex (6, 7), which is necessary for the initiation of SV40 DNA replication (7-9). RPA is also involved in the elongation phase of DNA replication, because it stimulates pol ␣, pol ␦, and pol ⑀ activity on a primed template DNA (10, 11).In nucleotide excision repair, RPA interacts with several key repair proteins, Xeroderma pigmentosum (XP) group A-complementing protein, XPA (12-15), XPG (13), and XPF-excision repair cross-complementation group 1 (16). RPA stabilizes the XPA-damaged DNA complex through the interaction with XPA, which appears to be essential for DNA repair (14, 17). RPA itself can interact with UV-damaged DNA (18); however, the physiological relevance of RPA-damaged DNA interaction in DNA repair is not clear. RPA is also involved in the later stage of nucleotide excision repair, gap-filling reaction, in collaboration with proliferating cell nuclear antigen, replication factor-C, and pol ␦ (or pol ⑀) (19). In homologous recombination, RPA physically interacts with Rad51 and Rad52, which appears to be essential for initiation of recombination (20 -24).The large subunit of RPA, p70, has multiple functional domains, including pol ␣ stimulation, ssDNA binding, and a conserved zinc finger domain with 4-cysteine type (3,5,25). The ssDNA binding domain of RP...

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In vitro analysis of the zinc-finger motif in human replication protein A

Cited by 26 publications

References 49 publications

Biochemical Analysis of the Damage Recognition Process in Nucleotide Excision Repair

Biochemical Analysis of the Damage Recognition Process in Nucleotide Excision Repair

Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Zinc Finger of Replication Protein A, a Non-DNA Binding Element, Regulates Its DNA Binding Activity through Redox

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