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PubMed2004

Recruitment of replication protein A by the papillomavirus E1 protein and modulation by single-stranded DNA.

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.

Binding, Competitive↗

PubMed2001

Damaged DNA-binding protein DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and replication protein A.

Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6-4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA + replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA + RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA.RPA, and thus stimulates the excision reaction of CPDs.

Base Sequence↗

PubMed2004

Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.

Arabidopsis↗

PubMed2004

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.

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr21-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr21 in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr21 was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr21 was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr21 phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr21 phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr21, whereas ETOP-induced Thr21 phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr21 phosphorylation by multiple DNA damage response protein kinases suggests that Thr21 phosphorylation of RPA32 is a crucial step within the DNA damage response.

Antibodies↗

PubMed1997

Dissociation of radiation-induced phosphorylation of replication protein A from the S-phase checkpoint.

Replication protein A (RPA) is a trimeric single-stranded DNA-binding protein complex involved in DNA replication, repair, and recombination. DNA damage induces phosphorylation of the RPA p34 subunit, and it has been speculated that this phosphorylation could contribute to the regulation of the DNA damage-induced S-phase checkpoint. To further examine this potential relationship, human cell lines expressing ataxia telangiectasia (AT)-mutated dominant-negative fragments, which fail to arrest in S phase in response to ionizing radiation (IR), and AT cells expressing AT-mutated-complementing fragments, which regain the ability to arrest replicative DNA synthesis in response to IR, were analyzed for radiation-induced RPA phosphorylation. Results from these studies demonstrate that IR-induced RPA phosphorylation can be uncoupled from the S-phase checkpoint, suggesting that RPA phosphorylation in response to IR is neither necessary nor sufficient for an S-phase arrest.

Cell Line, Transformed↗

PubMed1995

Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.

Amino Acid Sequence↗

PubMed2005

The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits.

Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.

Amino Acid Sequence↗

PubMed2005

Mass spectrometric identification of lysines involved in the interaction of human replication protein a with single-stranded DNA.

Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial cocrystal structure. 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. In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 DBD-AB-ssDNA complex but also revealed new protein contacts in the nucleoprotein complex. In addition to two residues, K263 and K343 of p70, previously identified by cocrystallography as direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA binding domain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259, are positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA interdomain structural reorganization induced by ssDNA binding.

Amino Acid Sequence↗

PubMed2001

Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIalpha and subunit IV of DNA polymerase alpha is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F.

Antigens, Neoplasm↗

PubMed1994

High-resolution genomic mapping of the three human replication protein A genes (RPA1, RPA2, and RPA3).

Human replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, homologous recombination, and excision repair. We have previously reported the cloning and bacterial overexpression of the three RPA genes and have mapped them to chromosome 1 (RPA2), chromosome 7 (RPA3), and chromosome 17 (RPA1). We have now obtained yeast strains with artificial chromosomes carrying the three human RPA genes and report the more detailed genomic mapping of RPA. RPA1 was mapped to chromosome 17p13.3 using a combination of PCR amplification of somatic cell hybrids and radiation hybrids containing chromosome 17 fragments. RPA2 was mapped to chromosome 1p35 by PCR amplification of somatic cell hybrids of chromosome 1 and by fluorescence in situ hybridization. RPA3 was mapped to chromosome 7p22 by Southern analysis and PCR amplification of somatic cell hybrids of chromosome 7 as well as fluorescence in situ hybridization. Since RPA is an essential component of major metabolic events affecting DNA, the physical mapping of the genes for it may help elucidate the biochemical basis of genetic disorders involving DNA metabolism.

Blotting, Southern↗

PubMed1992

Conservation of structure and function of DNA replication protein A in the trypanosomatid Crithidia fasciculata.

Human replication protein A (RP-A) is a three-subunit protein that is required for simian virus 40 (SV40) replication in vitro. The trypanosome homologue of RP-A has been purified from Crithidia fasciculata. It is a 1:1:1 complex of three polypeptides of 51, 28, and 14 kDa, binds single-stranded DNA via the large subunit, and is localized within the nucleus. C. fasciculata RP-A substitutes for human RP-A in the large tumor antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase alpha/primase, but it does not support efficient SV40 DNA replication in vitro. This extraordinary conservation of structure and function between human and trypanosome RP-A suggests that the mechanism of DNA replication, at both the initiation and the elongation level, is conserved in organisms that diverged from the main eukaryotic lineage very early in evolution.

Animals↗

PubMed1992

A geminivirus replication protein is a sequence-specific DNA binding protein.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.

Base Sequence↗

PubMed1997

Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA.

The single-stranded-DNA-binding proteins (SSBs) are essential for DNA function in prokaryotic and eukaryotic cells, mitochondria, phages and viruses. The structures of four SSBs have been solved, but the molecular details of the interaction of SSBs with DNA remain speculative. We report here the crystal structure at 2.4 A resolution of the single-stranded-DNA-binding domain of human replication protein A (RPA) bound to DNA. Replication protein A is a heterotrimeric SSB that is highly conserved in eukaryotes. The largest subunit, RPA70, binds to single-stranded (ss)DNA and mediates interactions with many cellular and viral proteins. The DNA-binding domain, which lies in the middle of RPA70, comprises two structurally homologous subdomains oriented in tandem. The ssDNA lies in a channel that extends from one subdomain to the other. The structure of each RPA70 subdomain is similar to those of the bacteriophage SSBs, indicating that the mechanism of ssDNA-binding is conserved.

Amino Acid Sequence↗

PubMed1999

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA.

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.

Bacterial Proteins↗

PubMed1997

Functional properties of replication fork assemblies established by the bacteriophage lambda O and P replication proteins.

We have used a set of bacteriophage lambda and Escherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of lambda replication forks. We demonstrate that the lambda replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650-750 nucleotides/s at 30 degrees C. This rate is identical to the fork movement rate we obtained using a minimal protein system, composed solely of E. coli DnaB helicase and DNA polymerase III holoenzyme. Our data are consistent with the conclusion that these two key bacterial replication proteins constitute the basic functional unit of a lambda replication fork. A comparison of rolling circle DNA replication in the minimal and lambda replication systems indicated that DNA synthesis proceeded for more extensive periods in the lambda system and produced longer DNA chains, which averaged nearly 200 kilobases in length. The higher potency of the lambda replication system is believed to result from its capacity to mediate efficient reloading of DnaB helicase onto rolling circle replication products, thereby permitting reinitiation of DNA chain elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually stimulated rolling circle DNA replication, but they apparently act indirectly by blocking accumulation of inhibitory free single-stranded DNA product. Finally, in the course of this work, we discovered that E. coli DNA polymerase III holoenzyme is itself capable of carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. coli single-stranded DNA-binding protein.

Bacterial Proteins↗

PubMed1997

Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A.

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.

Bacteriophage M13↗

PubMed2004

Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A.

Photochemical cross-linking of human replication protein A (hRPA) to oligonucleotide dT30 was performed to enable identification of amino acid sequences that reside in the DNA-binding domain. A nucleoprotein complex, with a 1:1 protein/DNA stoichiometry, was separated from unreacted enzyme and oligonucleotide by SDS-polyacrylamide gel electrophoresis and subjected to in-gel digestion with trypsin. Three cross-linked tryptic peptides (nucleopeptides) of hRPA70xdT30 (T43, T28/29, and a truncated T24/25) were isolated. Combined mass spectrometric and C-terminal proteolysis experiments showed that at least one amino acid in the segment 235-ATAFNE-240 (located in T24/25), at least one out of the two residues sequence 269-FT-270 (located in T28/29), and at least one from the sequence 383-VSDF-386 (located in T43) were involved in cross-linking. These peptides contained aromatic residues (F238, F269, and F386 respectively) that can form base-stacking interactions with the DNA and were, therefore, most likely to be involved in cross-linking. The results obtained in this study demonstrate that a combination of exhaustive proteolysis and MALDI TOF MS can localize the sites of DNA binding to very short sequences of amino acids. Data so acquired can confirm or amend information obtained from site-directed mutagenesis and X-ray crystallography.

Amino Acid Sequence↗

PubMed1983

Protein replication by amino acid pairing.

Biological problems concerning the origin of life and the mode of prion replication (Prusiner, 1982) may require protein replication (the synthesis of one protein sequence from another) as part of their solution. It is suggested that complementarity between protein sequences may be determined by amino acid pairing (Root-Bernstein, 1982a). Two mechanisms using the complementarity afforded by amino acid pairing are proposed. Experimental tests of the mechanisms are suggested.

Amino Acid Sequence↗