The role for zinc in Replication Protein A

Bochkareva, Elena; Korolev, Sergey; Bochkarev, A.

doi:10.1074/jbc.m000620200

Cited by 42 publications

(63 citation statements)

References 34 publications

(47 reference statements)

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“…These contacts and, in particular, the hydrogen bonds that the Asn496 and Lys497 backbone amide groups make to the phosphate group of Thy13 may facilitate the initial DBD-C-ssDNA association by stabilizing the transition from the loosely bound Thy10-Thy12 segment to the well-defined Thy13 at the groove entrance. Consistent with the Zn domain making a major contribution to DNAbinding, zinc-chelating agents dramatically reduce the affinity of DBD-C for DNA (Bochkareva et al 2000).…”

Section: Dbd-c-dna Contactsmentioning

confidence: 93%

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

2012

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Replication protein A (RPA) is the main eukaryotic ssDNA-binding protein with essential roles in DNA replication, recombination, and repair. RPA maintains the DNA as single-stranded and also interacts with other DNA-processing proteins, coordinating their assembly and disassembly on DNA. RPA binds to ssDNA in two conformational states with opposing affinities for DNA and proteins. The RPA-protein interactions are compatible with a low DNA affinity state that involves DNA-binding domain A (DBD-A) and DBD-B but not with the high DNA affinity state that additionally engages DBD-C and DBD-D. The structure of the high-affinity RPAssDNA complex reported here shows a compact quaternary structure held together by a four-way interface between DBD-B, DBD-C, the intervening linker (BC linker), and ssDNA. The BC linker binds into the DNAbinding groove of DBD-B, mimicking DNA. The associated conformational change and partial occlusion of the DBD-A-DBA-B protein-protein interaction site establish a mechanism for the allosteric coupling of RPA-DNA and RPA-protein interactions.

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Section: Dbd-c-dna Contactsmentioning

confidence: 93%

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

2012

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“…1A) (11, 56). SBD-A and SBD-B are structurally homologous as determined by X-ray crystallographic analysis of human RPA (hRPA), while the C-terminal domain (SBD-C) contains a C4-type zinc-finger motif (6,8,11). These three ssDNA binding domains share amino acid sequence similarity with each other and with the central region of Rpa2 (11).…”

mentioning

confidence: 99%

Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

Kim

Brill

2001

Molecular and Cellular Biology

114

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The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G 1 /S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G 2 /M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.Replication protein A (RPA) is a highly conserved eukaryotic single-stranded DNA (ssDNA) binding protein that was originally identified as a human factor required for simian virus 40 (SV40) DNA replication in vitro (19,84,86). As the eukaryotic equivalent of the Escherichia coli ssDNA binding protein (Ecssb), RPA plays multiple roles in DNA metabolism, including DNA replication, repair, and recombination (85). In addition to participating in both the initiation and elongation of DNA replication, RPA is required for nucleotide excision repair in vitro and physically interacts with XPA, XPG, and XPF (30,39,62,69). RPA is also required for genetic recombination and physically interacts with Rad52 and other factors (1,22,47,75).RPA is a heterotrimeric complex. In the budding yeast Saccharomyces cerevisiae it is composed of the Rpa1 (69 kDa), Rpa2 (36 kDa), and Rpa3 (13 kDa) subunits, which are encoded by RFA1, RFA2, and RFA3, respectively. Each subunit is essential for viability in yeast, and all three subunits are required for SV40 DNA replication in vitro (12,18,25,31,32). Rpa1 exhibits strong ssDNA binding activity on its own, and a subcomplex of Rpa2 and Rpa3 binds ssDNA weakly (7, 85). We have shown that yeast Rpa1 consists of four functional domains: an 18-kDa N-terminal domain that lacks ssDNA binding activity (Rpa1N) and three tandem ssDNA binding domains (SBDs; see Fig. 1A) (11, 56). SBD-A and SBD-B are structurally homologous as determined by X-ray crystallographic analysis of human RPA (hRPA), while the C-terminal domain (SBD-C) contains a C4-type zinc-finger motif (6,8,11). These three ssDNA binding domains share amino acid sequence similarity with each other and with the central region of Rpa2 (11). All fo...

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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).…”

mentioning

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 role for zinc in Replication Protein A

Cited by 42 publications

References 34 publications

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

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

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