Replication Protein A Complex Research Articles

Rad52 Protein Associates with Replication Protein A (RPA)-Single-stranded DNA to Accelerate Rad51-mediated Displacement of RPA and Presynaptic Complex Formation

The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDNA), but Rad52 protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 protein accelerates RPA displacement by Rad51 protein. This acceleration probably involves direct interactions with both Rad51 protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.

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.

Xeroderma Pigmentosum Complementation Group A Protein (XPA) Modulates RPA-DNA Interactions via Enhanced Complex Stability and Inhibition of Strand Separation Activity

Replication protein A (RPA) participates in many cellular functions including DNA replication and nucleotide excision repair. A direct interaction between RPA and the xeroderma pigmentosum group A protein (XPA) facilitates the assembly of a preincision complex during the processing of DNA damage by the nucleotide excision repair pathway. We demonstrate here the formation of a ternary RPA, XPA, and duplex cisplatin-damaged DNA complex as is evident by electrophoretic supershift analysis. The RPA-XPA complex displays modest specificity for damaged versus undamaged duplex DNA, and the RPA-XPA complex displays a greater affinity for binding duplex cisplatin-damaged DNA when compared with the RPA or XPA proteins alone, consistent with previous results. Using DNA denaturation assays, we demonstrate that the role of XPA is in the stabilization of the duplex DNA structure via inhibition of the strand separation activity of RPA. Rapid kinetic analysis indicates that the bimolecular k(on) of the RPA-XPA complex is 2.5-fold faster than RPA alone for binding a duplex cisplatin-damaged DNA. The dissociation rate, k(off), of the RPA-XPA complex is slower than that of the RPA protein alone, suggesting that the XPA protein stabilizes the initial binding of RPA to duplex DNA as well as maintaining the integrity of the duplex DNA. Interestingly, XPA has no effect on the k(on) of RPA for a single-stranded 40-mer DNA.

Replication Protein A in Pyrococcus furiosus Is Involved in Homologous DNA Recombination

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.

Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.

Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

Stability and Nuclear Distribution of Mammalian Replication Protein A Heterotrimeric Complex

Replication protein A (RPA), a stable complex of three polypeptides, is the single-stranded DNA-binding protein essential for DNA replication in eukaryotic cells. Previous studies of the subcellular distribution and stability of the RPA heterotrimer during the mammalian cell cycle have produced conflicting results. Here, we present evidence that these inconsistencies can be accounted for by the presence of an extractable pool of soluble RPA within the nucleus. Indirect immunofluorescence experiments in both CHO and HeLa cells showed that all three RPA subunits associated specifically with sites of ongoing DNA synthesis, similar to the replication fork protein proliferating cell nuclear antigen. Furthermore, we found no evidence for disassembly of the chromatin-bound heterotrimeric RPA complex in vivo. Our results are consistent with a role for RPA in the initiation and elongation steps of replication, as previously defined in the viral in vitro replication systems.

Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance

Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex . Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol α-primase. In addition, a delayed senescence phenotype was observed in rfa1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance.

Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A.

Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase alpha-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase alpha-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia coli as a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.

Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA.

Nondistorting C4' backbone adducts serve as molecular tools to analyze the strategy by which a limited number of human nucleotide excision repair (NER) factors recognize an infinite variety of DNA lesions. We have constructed composite DNA substrates containing a noncomplementary site adjacent to a nondistorting C4' adduct to show that the loss of hydrogen bonding contacts between partner strands is an essential signal for the recruitment of NER enzymes. This specific conformational requirement for excision is mediated by the affinity of xeroderma pigmentosum group A (XPA) protein for nonhybridizing sites in duplex DNA. XPA recognizes defective Watson-Crick base pair conformations even in the absence of DNA adducts or other covalent modifications, apparently through detection of hydrophobic base components that are abnormally exposed to the double helical surface. This recognition function of XPA is enhanced by replication protein A (RPA) such that, in combination, XPA and RPA constitute a potent molecular sensor of denatured base pairs. Our results indicate that the XPA-RPA complex may promote damage recognition by monitoring Watson-Crick base pair integrity, thereby recruiting the human NER system preferentially to sites where hybridization between complementary strands is weakened or entirely disrupted.

Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit.

Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding protein that is required for multiple processes in cellular DNA metabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates in multiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multiple domains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X2-C-X13-C-X2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate the roles of these domains in RPA function. The central DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part of the N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residues in RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these residues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.

Replication Protein A Induces the Unwinding of Long Double-stranded DNA Regions

We have investigated nucleoprotein filaments composed of human replication protein A (RPA) and DNA by electron microscopy. At low ionic strengths, RPA complexes with single-stranded DNA are similar in length to protein-free DNA suggesting that RPA-bound DNA remains in an extended configuration under these conditions. However, severe com paction of RPA-DNA complexes occurs in buffers with >2 mM MgCl 2or with 100 mM NaCl. At low ionic strengths, RPA binds to A + T-rich internal regions of linear double-stranded simian virus 40 (SV40) DNA and induces separation of complementary DNA strands. RPA also binds to closed- circular SV40 DNA, but requires the function of a DNA topoisomerase to invade and completely unwind duplex DNA regions. The ability of RPA to unwind long stretches of double-stranded DNA is not shared by the bacterial single-strand binding protein and the phage T4 gene 32 protein.

Dynamics of Human Replication Protein A Subunit Distribution and Partitioning in the Cell Cycle

Human replication protein A (RPA) is a three-subunit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In metaphase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.

Functional domains of the 70-kilodalton subunit of human replication protein A.

Human replication protein A (RPA) is a single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. This heterotrimeric complex is required for multiple processes in DNA metabolism including DNA replication, DNA repair, and recombination. Previous studies have suggested that the 616 amino acid, 70-kDa subunit of RPA (RPA 70) is composed of multiple structural/functional domains. We used a series of N-terminal deletions of RPA70 to define the boundaries of these domains and elucidate their functions. Mutant RPA complexes missing residues 1-168 of RPA70 bound ssDNA with high affinity and supported SV40 replication in vitro. In contrast, deletions extending beyond residue 168 showed a decreased affinity for ssDNA and were inactive in SV40 DNA replication. When residues 1-381 were deleted, the resulting truncated RPA70 was unable to bind ssDNA but still formed a stable complex with the 32- and 14-kDa subunits of RPA. Thus, the C-terminal domain of RPA70 is both necessary and sufficient for RPA complex formation. These data indicate that RPA70 is composed of three functional domains: an N-terminal domain that is not required for ssDNA binding or SV40 replication, a central DNA-binding domain, and a C-terminal domain that is essential for subunit interactions. For all mutant complexes examined, both phosphorylation of the 32-kDa subunit of RPA and the ability to support T antigen-dependent, origin-dependent DNA unwinding correlated with ssDNA binding activity.

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.

Structural Analysis of Human Replication Protein A: MAPPING FUNCTIONAL DOMAINS OF THE 70-kDa SUBUNIT

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is essential for DNA metabolism. Human RPA is composed of subunits of 70, 32, and 14 kDa with intrinsic DNA-binding activity localized to the 616-amino acid, 70-kDa subunit (RPA70). We have made a series of C-terminal deletions to map the functional domains of RPA70. Deletion of the C terminus resulted in polypeptides that were significantly more soluble than RPA70 but were unable to form stable complexes with the other two subunits of RPA. These data suggest that the C-terminal region of RPA70 may be important for complex formation. The DNA-binding domain was localized to a region of RPA70 between residues 1 and 441. A mutant containing residues 1-441 bound oligonucleotides with an intrinsic affinity close to wild-type RPA complex. This mutant also appeared to bind with reduced cooperativity. We conclude that the C terminus of RPA70 and the 32- and 14-kDa subunits are not involved directly with interactions with DNA but may have a role in cooperativity of RPA binding. RPA70 deletion mutants were not able to support DNA replication even in the presence of a complex of the 32- and 14-kDa subunits, suggesting that the heterotrimeric complex is essential for DNA replication. The putative zinc finger in the C terminus of RPA70 is not required for single-stranded DNA-binding activity.

DNA Repair Protein XPA Binds Replication Protein A (RPA)

XPA is a zinc finger DNA-binding protein, which is missing or altered in group A xeroderma pigmentosum cells and known to be involved in the damage-recognition step of the nucleotide excision repair (NER) processes. Using the yeast two-hybrid system to search for proteins that interact with XPA, we obtained the 34-kDa subunit of replication protein A (RPA, also known as HSSB and RFA). RPA is a stable complex of three polypeptides of 70, 34, 11 kDa and has been shown to be essential in the early steps of NER as well as in replication and recombination. We also demonstrate here that the RPA complex associates with XPA. These results suggest that RPA may cooperate with XPA in the early steps of the NER processes.

Recombinant replication protein A: expression, complex formation, and functional characterization

Replication protein A (RPA) is a multisubunit, single-stranded DNA-binding protein that is absolutely required for replication of SV40 DNA. The three cDNAs encoding the subunits of human replication protein A (70, 32, and 14 kDa) have been expressed individually and in combination in Escherichia coli. When subunits were expressed individually, appropriately sized polypeptides were synthesized, but were found to be either insoluble or aggregated with other proteins. We examined the interactions between individual RPA subunits by expressing pairs of subunits and determining if they formed stable complexes. Only the 32- and 14-kDa subunits formed a soluble complex when coexpressed. This complex was purified and characterized. The 32-14 kDa subcomplex did not have any effect on DNA replication and was not phosphorylated efficiently in vitro. We believe that the 32.14-kDa subcomplex may be a precursor in the assembly of the complete RPA complex. Coexpression of all three subunits of RPA resulted in a significant portion of each polypeptide forming a soluble complex. We have purified recombinant RPA complex from E. coli and demonstrated that it has properties similar to those of human RPA. Recombinant human RPA has the same subunit composition and the same activities as the authentic complex from human cells. Recombinant human RPA binds single-stranded DNA and is capable of supporting SV40 DNA replication in vitro. In addition, recombinant RPA became phosphorylated when incubated under replication conditions.

Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication

Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.