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PubMed1999

Enhancement of UV-induced cytotoxicity by the adeno-associated virus replication proteins.

Adeno-associated virus (AAV) normally requires co-infection of a helper virus to complete its life cycle. However, under conditions of cellular stress, such as treatment with carcinogens or ultraviolet (UV) light, a permissive intracellular environment is established and AAV completes its replicative cycle producing low levels of progeny virus. AAV DNA replication is dependent upon viral replication proteins, Rep78 and Rep68. The detailed mechanism by which these proteins interact with host cell factors is unknown. We have used a cell line (Neo6) that inducibly expresses the AAV Rep proteins to study their effects on cells that have undergone UV-induced DNA damage. Induction of Rep protein expression immediately after a sub-lethal dose of UV irradiation resulted in rapid cell killing. Those cells that die had chromatin condensation while cellular membranes remained intact, suggesting that concurrent Rep expression and UV damage induces an apoptosis-like response. However, we did not observe any DNA degradation. Thus we believe that the combination of Rep expression and UV irradiation induces cell death that shares some of the characteristics of apoptosis. UV irradiation and Rep expression induced an increase in the level of the CDK inhibitor, p21Cip, and the appearance of modified forms of both p21Cip and Bcl-2. Alteration of normal expression of these cytostatic/apoptotic proteins provides insight into the intracellular targets of the AAV replication proteins.

Apoptosis↗
PubMed2006

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda.

DNA polymerase lambda (pol lambda) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol lambda either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol lambda had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol lambda; (iii) pol lambda preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol lambda, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol lambda and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol lambda are discussed.

Base Pair Mismatch↗
PubMed2000

Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA.

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.

Conserved Sequence↗
PubMed1996

Interactions between geminivirus replication proteins.

Geminiviruses are small DNA viruses that replicate in the nuclei of infected plant cells. The closely related geminiviruses tomato golden mosaic virus and bean golden mosaic virus each encode a protein, AL1, that catalyzes the initiation of rolling-circle replication. Both viruses also specify a second replication protein, AL3, that greatly enhances the level of viral DNA accumulation. Using recombinant proteins produced in a baculovirus expression system, we showed that AL1 copurifies with a protein fusion of glutathione S-transferase (GST) and AL1, independent of the GST domain. Similarly, authentic AL3 cofractionates with a GST-AL3 fusion protein. These results demonstrated that both AL1 and AL3 form oligomers. Immunoprecipitation of protein extracts from insect cells expressing both AL1 and AL3 showed that the two proteins also complex with each other. None of the protein interactions displayed virus specificity; the tomato and bean golden mosaic virus proteins complexed with each other. The addition of heterologous replication proteins had no effect on the efficiency of geminivirus replication in transient-replication assays, suggesting that heteroprotein complexes might be functional. The significance of these protein interactions is discussed with respect to geminivirus replication in plant cells.

Geminiviridae↗
PubMed1993

The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.

Ataxia Telangiectasia↗
PubMed2004

Physical interaction between replication protein A and Rad51 promotes exchange on single-stranded DNA.

Replication protein A (RPA) is displaced from single-stranded DNA (ssDNA) by Rad51 during the initiation of homologous recombination. Interactions between these proteins have been reported, but the functional significance of the direct RPA-Rad51 interaction has yet to be elucidated. We have identified and characterized the interaction between DNA-binding domain A of RPA (RPA70A) and the N-terminal domain of Rad51 (Rad51N). NMR chemical shift mapping showed that Rad51N binds to the ssDNA-binding site of RPA70A, suggesting a competitive mechanism for the displacement of RPA from ssDNA by Rad51. A structure of the RPA70A-Rad51N complex was generated by experimentally guided modeling and then used to design mutations that disrupt the binding interface. Functional ATP hydrolysis assays were performed for wild-type Rad51 and a mutant defective in binding RPA. Rates of RPA displacement for the mutant were significantly below those of wild-type Rad51, suggesting that a direct RPA-Rad51 interaction is involved in displacing RPA in the initiation stage of genetic recombination.

Adenosine Triphosphate↗
PubMed1998

Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases.

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.

Azides↗
PubMed2002

Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation.

Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. We also demonstrate that Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction.

Adenosine Triphosphatases↗
PubMed2006

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases.

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of 35S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70-RPA32-RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.

Antibodies, Antinuclear↗
PubMed2004

[Prokaryotic expression and immuno-localization of replication protein gene of Tobacco curly shoot virus].

The replication protein (Rep) gene of Tobacco curly shoot virus (TbCSV) Y35 isolate was obtained from the infected tobacco plants by PCR and cloned into expression vector pGEX-4T-1 to generate the recombinant plasmid pGEX-Y35Rep. The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLys S, and TbCSV Rep fusion protein was expressed with induction by IPTG and some products were soluble. The Rep fusion protein was purified with GST-Sepharose 4B affinity chromatography and its polyclonal antibody was produced in a rabbit. Studies on subcellular distribution of TbCSV Rep protein in infected tobacco leaves revealed that Rep protein mainly exists in the fractions containing the nucleus. Immuno-gold labeling with the antibody against Rep fusion protein also indicated that Rep protein localized in the nuclei of infected tobacco cells.

Begomovirus↗
PubMed1996

Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA.

Replication protein A (RPA) is multisubunit single-stranded DNA-binding protein required for multiple processes in DNA metabolism including DNA replication, DNA repair, and recombination. Human RPA is a stable complex of three subunits of 70, 32, and 14 kDa (RPA70, RPA32, and RPA14, respectively). We examined the structure of both wild-type and mutant forms of human RPA by mapping sites sensitive to proteolytic cleavage. For all three subunits, only a subset of the possible protease cleavage sites was sensitive to digestion. RPA70 was cleaved into multiple fragments of defined lengths. RPA32 was cleaved rapidly to a approximately 28-kDa polypeptide containing the C-terminus that was partially resistant to further digestion. RPA14 was refractory to digestion under the conditions used in these studies. The digestion properties of a complex of RPA32 and RPA14 were similar to those of the native heterotrimeric RPA complex, indicating that the structure of these subunits is similar in both complexes. Epitopes recognized by monoclonal antibodies to RPA70 were mapped, and this information was used to determine the position of individual cleavage events. These studies suggest that RPA70 is composed of at least two structural domains: an approximately 18-kDa N-terminal domain and a approximately 52-kDa C-terminal domain. The N-terminus of RPA70 was not required for single-stranded DNA-binding activity. Multiple changes in the digestion pattern were observed when RPA bound single-stranded DNA: degradation of the approximately 52-kDa domain of RPA70 was inhibited while proteolysis of RPA32 was stimulated. These data indicate that RPA undergoes a conformational change upon binding to single-stranded DNA.

Antibodies, Monoclonal↗
PubMed2006

Replication-dependent DNA damage response triggered by roscovitine induces an uncoupling of DNA replication proteins.

The cyclin-dependent kinase (CDK) inhibitor roscovitine is under evaluation in clinical trials for its antiproliferative properties. Roscovitine arrests cell cycle progression in G(1) and in G(2) phase by inhibiting CDK2 and CDK1, and possibly CDK7 and CDK9. However, the effects of CDK2 inhibition in S-phase cells have been not fully investigated. Here, we show that a short-term treatment with roscovitine is sufficient to inhibit DNA synthesis, and to activate a DNA damage checkpoint response, as indicated by phosphorylation of p53-Ser15, replication protein A, and histone H2AX. Analysis of DNA replication proteins loaded onto DNA during S phase showed that the amount of proliferating cell nuclear antigen (PCNA), a cofactor of DNA replication enzymes, was significantly reduced by roscovitine. In contrast, chromatin-bound levels of DNA polymerase delta, DNA ligase I and CDK2, were stabilized. Checkpoint inhibition with caffeine could rescue PCNA disassembly only partially, pointing to additional effects due to CDK2 inhibition and the presence of replication stress. These results suggest that in S-phase cells, roscovitine induces checkpoint-dependent and -independent effects, leading to stabilization of replication forks and an uncoupling between PCNA and PCNA-interacting proteins.

Cell Proliferation↗
PubMed2003

Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro.

We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae. The stimulation of Dna2 endonuclease activity by RPA requires direct protein-protein interaction. In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA. RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation. In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1. Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins. Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity. We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro. The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed.

Adenosine Triphosphatases↗
PubMed2005

DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage.

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

Amino Acid Sequence↗
PubMed1998

Replication protein A stimulates long patch DNA base excision repair.

Two pathways for completion of DNA base excision repair (BER) have recently emerged. In one, called short patch BER, only the damaged nucleotide is replaced, whereas in the second, known as long patch BER, the monobasic lesion is removed along with additional downstream nucleotides. Flap endonuclease 1, which preferentially cleaves unannealed 5'-flap structures in DNA, has been shown to play a crucial role in the long patch mode of repair. This nuclease will efficiently release 5'-terminal abasic lesions as part of an intact oligonucleotide when cleavage is combined with strand displacement synthesis. Further gap filling and ligation complete repair. We reconstituted the final steps of long patch base excision repair in vitro using calf DNA polymerase epsilon to provide strand displacement synthesis, human flap endonuclease 1, and human DNA ligase I. Replication protein A is an important constituent of the DNA replication machinery. It also has been shown to interact with an early component of base excision repair: uracil glycosylase. Here we show that human replication protein A greatly stimulates long patch base excision repair.

DNA Ligase ATP↗
PubMed1999

Phosphorylation of replication protein A middle subunit (RPA32) leads to a disassembly of the RPA heterotrimer.

Replication protein A (RPA), the major eukaryotic single-strand specific DNA binding protein, consists of three subunits, RPA70, RPA32, and RPA14. The middle subunit, RPA32, is phosphorylated in a cell cycle-dependent manner. RPA occurs in two nuclear compartments, bound to chromatin or free in the nucleosol. We show here that the chromatin-associated fraction of RPA contains the phosphorylated forms of RPA32. Treatment of chromatin with 0.4 M NaCl releases bound RPA and causes a separation of the large and the phosphorylated middle RPA subunit. Unmodified RPA in the nucleosolic fraction remains perfectly stable under identical conditions. Phosphorylation is most likely an important determinant of RPA desintegration because dialysis from 0.4 to 0.1 NaCl causes the reformation of trimeric RPA only under dephosphorylating conditions. Biochemical studies with isolated Cyclin-dependent protein kinases showed that cyclin A/CDK1 and cyclin B/CDK1, but not cyclin E/CDK2, can phosphorylate human recombinant RPA in vitro. However, only a small fraction of in vitro phosphorylated RPA desintegrated, suggesting that phosphorylation may be one, but probably not the only, determinant affecting subunit interaction. We speculate that phosphorylation and changes in subunit interaction are required for the proposed role of RPA during the polymerase switch at replication forks.

CDC2 Protein Kinase↗
PubMed2004

Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks.

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.

Acid Anhydride Hydrolases↗