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PubMed2000

5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates.

Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) "pseudo-origin" (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cis-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin.

3' Untranslated Regions↗

PubMed2003

Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2.

Mutations in the BRCA1 and BRCA2 genes predispose women to familial, early-onset breast cancer. Both the BRCA1 and BRCA2 proteins appear to function in the homologous recombination pathway of DNA double-strand break repair. Both BRCA1 and BRCA2 have also been implicated in transcription by RNA polymerase II, for both proteins have domains which, when tethered adjacent to a promoter, can activate transcription. In experiments reported here, we have used protein affinity chromatography and coimmunoprecipitation techniques to show that the putative N-terminal acidic transcriptional activation domain of BRCA2 interacts with replication protein A (RPA), a protein essential for DNA repair, replication and recombination. This interaction was not mediated by DNA and was specific for human RPA but not yeast RPA. Since the cancer-predisposing mutation Y42C in BRCA2 significantly compromised the interaction between RPA and BRCA2, this interaction may be biologically important. That BRCA2 protein in HeLa cell extract also coimmunoprecipitated with RPA suggested that this interaction occurs in vivo. Therefore, the transcriptional activation domains within BRCA2, and perhaps BRCA1, may provide links to RPA and DNA repair processes rather than transcription.

BRCA2 Protein↗

PubMed1986

The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA.

The in vitro replication of R1 miniplasmid promoted by purified preparations of the plasmid encoded RepA protein in cell extracts of E. coli is resistant to rifampicin and can be completely inhibited by antibodies against DnaG, the primase of the cell, as well as by antibodies against proteins DnaB and SSB. R1 replication is abolished in extracts deficient in the DnaA protein. This deficiency is efficiently complemented by purified preparations of the DnaA protein. The in vitro replication of plasmid R1 is also abolished in DnaC deficient extracts and by a 10 bp deletion (nucleotides 1463-1472) within the minimal origin region. These data indicate the requirement of the DnaA, DnaB, DnaC, DnaG and SSB replication proteins of the host, as well as of specific oriR1 sequences for the RepA dependent replication of plasmid R1. The implications of these results for the initiation of R1 replication are discussed.

Bacterial Proteins↗

PubMed1999

The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.

Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of RPA32 and the entire RPA14 subunit. The structure revealed that RPA14 and the central part of RPA32 are structural homologues. Each subunit contains a central OB-fold domain, which also resembles the DNA-binding domains in RPA70; an N-terminal extension that interacts with the central OB-fold domain; and a C-terminal helix that mediate heterodimerization via a helix-helix interaction. The OB-fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA-binding domains, supporting a DNA-binding role for RPA32. The discovery of a third and fourth OB-fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB-folds, is conserved in evolution. The structure also suggests a mechanism for RPA trimer formation.

Amino Acid Sequence↗

PubMed2005

53BP1 is associated with replication protein A and is required for RPA2 hyperphosphorylation following DNA damage.

p53-binding protein 1 (53BP1) acts as an 'adaptor/mediator' for transducing DNA damage signals, especially following detection of DNA double-strand breaks. In an effort to broaden our understanding of the protein network surrounding 53BP1, we isolated possible 53BP1 binding partners by co-immunoprecipitation, and identified them via tandem mass spectrometric analysis. The 53BP1-associated proteins included RPA1 and RPA2, two components of the replication protein A (RPA) complex. The presence of RPA components in the immunoprecipitates was confirmed by immunoblotting, and we found that the association between 53BP1 and RPA2 was disrupted following DNA damage induced by treatment with camptothecin, a topoisomerase I inhibitor. To investigate the functional meaning of the 53BP1 and RPA interaction, we established U2OS osteosarcoma cell lines stably expressing dominant-negative fragments of 53BP1. We found that camptothecin-induced RPA2 phosphorylation was inhibited in these cells, and also following 53BP1 knockdown by siRNA transfection. On the cellular level, camptothecin-induced apoptosis was augmented in the dominant-negative cell lines, resulting in increased chemosensitivity to this drug. Taken together, these results suggest that 53BP1 is involved in DNA damage-induced RPA2 hyperphosphorylation, and inhibition of 53BP1 function may sensitize cancer cells to camptothecin treatment.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Cloning and characterization of replication protein A p32 complementary DNA in zebrafish (Danio rerio).

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (70, 32, and 14 kDa) that is an essential component of the DNA replication fork. A complementary DNA encoding zebrafish RPA 32-kDa subunit was isolated by screening a zebrafish embryo lambda APII cDNA library with a human RPA p32 cDNA probe. The zebrafish RPA p32 cDNA consisted of 1097 bp encoding 272 amino acid residues. The deduced amino acid sequence shows high similarity to mouse and human RPA p32. In vitro phosphorylation of zebrafish RPA protein by Cdc2 kinase was shown. A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography. In this article, cloning of the zebrafish RPA p32 cDNA is reported in relation to the study of DNA replication in fish.

Journal Article↗

PubMed1997

Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner.

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor suppressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.

Animals↗

PubMed1998

The 32- and 14-kilodalton subunits of replication protein A are responsible for species-specific interactions with single-stranded DNA.

Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.

Binding Sites↗

PubMed2003

The 32-kilodalton subunit of replication protein A interacts with menin, the product of the MEN1 tumor suppressor gene.

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.

Animals↗

PubMed1986

The Escherichia coli dnaB replication protein is a DNA helicase.

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.

Antibodies↗

PubMed2002

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.

Crossing Over, Genetic↗

PubMed2003

Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.

Arabidopsis↗

PubMed1999

Replication protein A (RPA) binding to duplex cisplatin-damaged DNA is mediated through the generation of single-stranded DNA.

Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808-8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5-2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3-4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.

Cisplatin↗

PubMed1983

In vitro complementation as an assay for purification of adenovirus DNA replication proteins.

As an approach to the purification of adenovirus-encoded DNA replication proteins, we have developed in vitro complementation assays that make use of viral mutants defective in DNA replication in vivo. Nuclear extracts prepared from cells infected with H5ts36 or H5ts125, two such mutants belonging to different complementation groups, were found to be defective in viral DNA replication in vitro. However, replication activity could be restored by mixing the two extracts. Replication activity in either extract also could be restored by addition of appropriate replication-deficient fractions purified from cells infected with wild-type adenovirus. By using such assays, H5ts36- and H5ts125-complementing activities were extensively purified. As expected, purified H5ts125-complementing activity consisted of a single major polypeptide, the 72-kilodalton (kDal) adenovirus DNA binding protein. The purified H5ts36-complementing activity consisted of the 80-kDal adenovirus terminal protein precursor and two other major polypeptides with apparent molecular masses of 140 and 65 kDal. Formation of the 80-kDal terminal protein-dCMP complexes, the proposed initial step in adenovirus DNA replication, required components in the purified H5ts36-complementing fraction and a cellular factor(s) but did not require the adenovirus DNA binding protein. The complete in vitro adenovirus DNA replication reaction was reconstituted from the purified H5ts36-complementing activity, the adenovirus DNA binding protein, and an extract from uninfected cells.

Adenoviruses, Human↗

PubMed1999

The plasmid ColIb-P9 antisense Inc RNA controls expression of the RepZ replication protein and its positive regulator repY with different mechanisms.

The autonomous replication region of plasmid ColIb-P9 contains repZ encoding the RepZ replication protein, and inc and repY as the negative and positive regulators of repZ translation, respectively. inc encodes the antisense Inc RNA, and repY is a short open reading frame upstream of repZ. Translation of repY enables repZ translation by inducing formation of a pseudoknot containing stem-loop I, which base pairs with the sequence preceding the repZ start codon. Inc RNA inhibits both repY translation and formation of the pseudoknot by binding to the loop I. To investigate control of repY expression by Inc RNA, we isolated a number of mutations that express repY in the presence of Inc RNA. One class of mutations delete a part of another stem-loop (II), which derepresses repY expression by initiating translation at codon 10 (GUG), located within this structure. Point mutations in stem-loop II can also derepress repY translation, and the introduction of compensatory base-changes restores control of repY translation. These results not only indicate that suppressing a cryptic start codon by secondary structure is important for maintaining the translational control of repZ but also demonstrate that the position of start site for repY translation is critical for its control by Inc RNA. Thus, Inc RNA controls repY translation by binding in the vicinity of the start codon, in contrast to the control of repZ expression at the level of loop-loop interaction.

Bacterial Proteins↗

PubMed2003

Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface.

Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.

Conserved Sequence↗

PubMed2003

The herpes simplex virus (HSV) protein ICP34.5 is a virion component that forms a DNA-binding complex with proliferating cell nuclear antigen and HSV replication proteins.

The replicative ability of ICP34.5-null herpes simplex virus (HSV) is cell type and state dependent. In certain cells, ICP34.5 interacts with protein phosphatase 1 to preclude host cell protein synthesis shutoff by dephosphorylation of the eukaryotic initiation factor eIF-2alpha. However, host cell shutoff is not induced by ICP34.5-null HSV in most cells, irrespective of type and state. In general, dividing cells support replication of ICP34.5-null HSV; nondividing cells cannot. Previously the authors showed that ICP34.5 binds to proliferating cell nuclear antigen (PCNA), a protein necessary for cellular DNA replication and repair. Here the authors demonstrate that (1) the interaction between ICP34.5 and PCNA involves two regions of the virus protein; (2) ICP34.5 forms a complex with HSV replication proteins that is DNA binding; (3) at early times in infection, ICP34.5 colocalizes with PCNA and HSV replication proteins in cell nuclei, before accumulating in the cytoplasm; and (4) ICP34.5 is a virion protein. In light of ongoing clinical trials assessing the safety and efficacy of ICP34.5-null HSV, it is vital that the roles of ICP34.5 in HSV replication are understood. The authors propose that in nondividing cells, ICP34.5 is required to switch PCNA from repair to replication mode, a prerequisite for the initiation of HSV replication.

Animals↗

PubMed2002

Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer.

PURPOSE: We sought to identify autoantigens recognized by antibodies in breast cancer patient sera with potential diagnostic or prognostic significance. EXPERIMENTAL DESIGN: Serum from a female breast cancer patient exhibiting a high titer antinuclear antibody was used to screen a HeLa cDNA expression library, leading to the cloning of a cDNA for the M(r) 32,000 subunit of replication protein A (RPA32). RPA32 expression and localization were assayed in autologous tumor by monoclonal antibody staining. A specific ELISA using recombinant protein was used to screen sera from 801 breast cancer patients and 65 controls. RESULTS: A relationship between anti-replication protein A (RPA) antibodies and the ductal breast carcinoma of the proband was suggested by overexpression and aberrant localization of RPA32 in tumor cells as compared with surrounding normal ductal tissue and by the presence of anti-RPA32 antibodies before the diagnosis. The prevalence of anti-RPA32 antibodies was significantly higher (P < 0.01) among breast cancer patients (87 of 801 patients) than among noncancer controls (0 of 65 controls). Similarly, anti-RPA32 antibodies were present in 4 of 39 patients with intraductal in situ carcinoma. No associations were found between anti-RPA antibodies and survival, occurrence of a second tumor, metastases, or antibodies to p53. Reactivity to RPA32 also was detected in sera from 3 of 47 patients with other cancers. CONCLUSIONS: In view of the central role of RPA in DNA replication, recombination, and repair, we suggest that autoimmunity to RPA32 may reflect molecular changes involved in the process of tumorigenesis. The finding of antibodies to RPA32 before diagnosis and their prevalence in in situ carcinoma suggest that they are potentially useful markers of early disease.

Antigens, Neoplasm↗