HIV-1 nucleocapsid protein and replication protein A influence the strand displacement DNA synthesis of lentiviral reverse transcriptase.
Explore the source record for details and available documents.
Animals↗
SEARCH · Search PubMed
Results for “Replication Protein A”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains.
The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein-DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5'-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a 'zero-length' photoreactive group (4-thio-2'-deoxyuridine-5'-monophosphate) embedded in the 3' end of the DNA primer, or a group connected to the 3' end by a lengthy linker (5-[N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C.p32D.p14) was capable of correctly recognizing the primer- template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D.p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process.
Base Sequence↗
Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A.
One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.
Amino Acid Sequence↗
Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA.
The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.
Amino Acid Sequence↗
The role for zinc in replication protein A.
Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. The proteolytic core of this domain comprised amino acids 432-616. X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (amino acids 43-171), and RPA14 had strong DNA binding activity. When properly coordinated with zinc, the trimer's affinity to ssDNA was only 3-10-fold less than that of the ssDNA-binding domain in the middle of RPA70. However, the DNA-binding activity of the trimer was dramatically reduced in the presence of chelating agents. Our data indicate that (i) Zn(II) is essential to stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses DNA-binding activity, which is modulated by Zn(II); and (iii) ssDNA binding by the trimer is a synergistic effect generated by the RPA70-CTD and RPA32.
Amino Acid Sequence↗
Development of a high-throughput screen for inhibitors of replication protein A and its role in nucleotide excision repair.
The heterotrimeric protein, replication protein A (RPA), is essential for DNA repair and replication. RPA is a viable target in the treatment of cancer as many chemotherapeutic agents act by blocking DNA replication. Furthermore, inhibition of RPA could prove useful in treating cancers that have acquired resistance to DNA damaging agents through enhanced DNA repair mechanisms as has been observed with certain platinum-resistant carcinomas. In an effort to identify inhibitors of RPA, we employed a novel fluorescent reporter and established a homogeneous high-throughput screening assay to measure RPA's DNA binding activity. Using this assay, we have screened a collection of small molecules and determined the effect they have on the RPA-DNA interaction. Of the 2000 compounds screened, 79 scored positive for inhibition of RPA binding activity. Secondary screenings were performed using an electrophoretic mobility shift assay; of the 79 compounds, 9 scored positive and were further characterized in titration experiments to determine the most potent inhibitor, resulting in several compounds showing an IC50 in the low micromolar range. Fluorescence polarization analyses were also performed to determine the mechanism of inhibition for each compound. Validation of the inhibitory activity of selected compounds was verified using in vitro nucleotide excision repair (NER) catalyzed excision of a single cisplatin lesion in a duplex DNA. The identification and use of RPA inhibitors may aid in inhibiting NER activity that could potentially circumvent resistance to certain chemotherapeutic agents as well as be useful in the characterization of RPA and its interaction with DNA.
Antineoplastic Agents↗
Replication protein A is sequentially phosphorylated during meiosis.
Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress. In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses. We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis. Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1. In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1. While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete. Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks. Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression.
DNA Replication↗
Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.
The purified human single-stranded DNA binding protein, replication protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique. RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase. The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase. Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase. These results suggest that the specific protein-protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA replication.
Amino Acid Sequence↗
Physical and functional mapping of the replication protein a interaction domain of the werner and bloom syndrome helicases.
The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN(H-R)) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN(H-R) helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN(H-R) had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM(642-1290), that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.
Adenosine Triphosphatases↗
DNA stimulates Mec1-mediated phosphorylation of replication protein A.
The cellular single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) becomes phosphorylated periodically during the normal cell cycle and also in response to DNA damage. In Saccharomyces cerevisiae, RPA phosphorylation requires the checkpoint protein Mec1, a protein kinase homologous in structure and function to human ATR. We confirm here that immunocomplexes containing a tagged version of Mec1 catalyze phosphorylation of purified RPA, likely reflecting an RPA kinase activity intrinsic to Mec1. A significant stimulation of this activity is observed upon the addition of covalently closed ssDNA derived from the bacteriophage M13. This stimulation is not observed with mutant RPA deficient for DNA binding, indicating that DNA-bound RPA is a preferred substrate. Stimulation is also observed upon the addition of linear ssDNA homopolymers or hydrolyzed M13 ssDNA. In contrast to circular ssDNA, these DNA cofactors stimulate both wild type and mutant RPA phosphorylation. This finding suggests that linear ssDNA can also stimulate Mec1-mediated RPA phosphorylation by activating Mec1 or an associated protein. Although the Mec1-interacting protein Ddc2 is required for RPA phosphorylation in vivo, it is required for neither basal nor ssDNA-stimulated RPA phosphorylation in vitro. Therefore, activation of Mec1-mediated RPA phosphorylation by either circular or linear ssDNA does not operate through Ddc2. Our results provide insight into the mechanisms that function in vivo to specifically induce RPA phosphorylation upon initiation of DNA replication, repair, or recombination.
Bacteriophages↗
Phosphorylation of replication protein A by S-phase checkpoint kinases.
The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3'-, but not 5'-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3'-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3'-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.
Ataxia Telangiectasia Mutated Proteins↗
The binding-site sizes of Escherichia coli single-stranded-DNA-binding protein and mammalian replication protein A are 65 and >/= 54 nucleotides respectively.
The electrophoretic mobilities of complexes formed with single-stranded (ss) DNA and tetrameric Escherichia coli ssDNA-binding protein (EcoSSB) or mammalian replication protein A (RPA) were analysed. The electrophoretic mobilities of the complexes in a native polyacrylamide gel increased as the lengths of the DNA increased from 28 to 70 nt, thus revealing paradoxical 'descending-staircase' patterns. Increases in the electrophoretic mobilities of EcoSSB.ssDNA complexes were observed when the lengths of the bound DNA were increased by 1 nt. Quantitative analyses of the complexes suggested that the binding-sites sizes of EcoSSB and RPA were 65 and >=54 nt respectively. The binding-site size for RPA is at least 24 nt larger than previously reported.
Animals↗
Geminivirus C3 protein: replication enhancement and protein interactions.
Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.
Amino Acid Sequence↗
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.
Base Sequence↗
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.
Adenosine Triphosphatases↗
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.
Amino Acid Sequence↗
Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro.
Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo. T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer. Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein. 59 protein slows replication without the helicase. As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase. A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences.
Bacteriophage T4↗
Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.
Replication protein A (RPA, also known as human single-stranded DNA-binding protein) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other DNA-damaging agents, which implicates the modified protein in the regulation of DNA replication after DNA damage or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the protein. In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the protein from DNA replication to DNA repair.
Androstadienes↗