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At least 19 recordsLinked to original sources
PubMed1995

Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH.

Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.

2-Acetylaminofluorene↗
PubMed1996

Dissection of functional domains of the human DNA replication protein complex replication protein A.

Replication protein A (RPA) is a mammalian single-stranded DNA binding factor essential for DNA replication, repair, and recombination. It is composed of three subunits of 70, 34, and 13 kDa (Rpa1, Rpa2, and Rpa3, respectively). Deletion mapping of the Rpa2 subunit identified the domain required for interaction with Rpa1 and Rpa3 which does not include the N-terminal domain that is phosphorylated during S phase. Deletion mapping of Rpa1 defined three domains. The C-terminal third of the Rpa1 polypeptide binds Rpa2 which itself forms a bridge between Rpa1 and Rpa3. The N-terminal third of Rpa1 bound single-stranded DNA under low stringency conditions only (0.1 M NaCl), while a central domain binds to single-stranded DNA under both low and high stringency conditions (0.5 M NaCl). Binding to p53 requires the N-terminal third of Rpa1 with some contribution from the C-terminal third. The evolutionarily conserved putative zinc finger near the C terminus of Rpa1 was not required for binding to single-stranded DNA, Rpa2, or p53. However, all three subdomains of Rpa1 and the zinc finger were essential for supporting DNA replication in vitro. These experiments are a first step toward defining peptide components responsible for the many functions of the RPA protein complex.

Animals↗
PubMed1998

Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A.

Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.

Amino Acid Sequence↗
PubMed1993

Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A.

Replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, recombination, and repair. We have previously reported the cloning and bacterial expression of the 70- and 32-kDa subunits of human RPA (hRPA). We have now cloned the 14-kDa subunit (hRPA3) from a HeLa cell cDNA library. The hRPA3 cDNA is a 692-base pair sequence that contains an open reading frame encoding a protein of 121 amino acids with a calculated molecular mass of 13.6 kDa. The deduced amino acid sequence shows only limited similarity to the small subunit of yeast RPA and is unrelated to any other protein in the current data banks. A recombinant protein containing a short histidine tag at the NH2 terminus has been purified in good yield from Escherichia coli by metal-chelate affinity chromatography. Antibodies prepared against recombinant hRPA3 recognize the native protein and inhibit SV40 DNA replication in vitro. We have localized the genes for the 70-, 32-, and 14-kDa subunits to chromosomes 17, 1, and 7, respectively, using polymerase chain reaction amplification of genomic DNA from rodent-human hybrid cell lines. Since RPA appears to be involved in several fundamental cellular processes, the physical mapping of the RPA genes may be useful in identifying possible human genetic defects associated with RPA deficiency or dysfunction.

Amino Acid Sequence↗
PubMed1996

A hierarchy of SSB protomers in replication protein A.

Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.

Amino Acid Sequence↗
PubMed1992

DNA unwinding activity of replication protein A.

Replication protein A (RP-A) is a heterotrimeric complex conserved in eukaryotic cells. It binds to single-stranded DNA and is essential for initiation and elongation of DNA replication. In this communication we give evidence that this protein can unwind DNA independent of magnesium and ATP, two essential cofactors for bona fide DNA helicase activity. RP-A can unwind up to at least 350 basepairs and appears to be required in stoichiometric amounts. The reaction is extremely sensitive to NaCl and MgCl2. This activity of RF-A is suggestive for a possible unwinding function in initiation of DNA replication in eukaryotes.

Adenoviridae↗
PubMed1998

Chromatin association of replication protein A.

Replication protein A (RPA) is the major single strand-specific DNA-binding protein in eukaryotic cells. We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Single strand-specific endonucleases failed to mobilize RPA bound to chromatin in G1 phase and S phase HeLa cells. In contrast, brief treatments with pancreatic DNase I or with micrococcal nuclease sufficed to release RPA from its chromatin-binding sites. Sucrose gradient analysis of soluble micrococcal nuclease digests showed that the released RPA sedimented free of mono- or oligonucleosomal chromatin fragments, possibly indicating that most of the detectable RPA may be associated with chromatin sites, which are more open to nuclease attack than bulk chromatin. The surprising conclusion is that the majority of the detectable RPA is, either directly or indirectly, associated with double-stranded DNA regions in chromatin from HeLa cells in G1 phase and in S phase.

Binding Sites↗
PubMed1997

The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

Alleles↗
PubMed1990

The primary structure of the 32-kDa subunit of human replication protein A.

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.

Amino Acid Sequence↗
PubMed1984

Initiation of DNA synthesis on single-stranded DNA templates in vitro promoted by the bacteriophage lambda O and P replication proteins.

The bacteriophage lambda O and P protein replication initiators, in conjunction with six purified Escherichia coli replication proteins, replicate the single-stranded chromosomes of phages M13 and phi X174 to a duplex form. Several discrete steps are involved in this DNA synthesis reaction. In an ATP-dependent step that precedes priming, the lambda O and P proteins interact with the Escherichia coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto DNA coated with single-stranded DNA binding protein. This creates a stable prepriming intermediate, isolable by gel filtration, that is rapidly primed and replicated upon the addition of primase and DNA polymerase III holoenzyme. Each of the eight proteins required for this nonspecific single strand replication reaction also have physiological roles in the replication of the bacteriophage lambda chromosome in vivo. We propose a scheme for the lambda O and P protein-dependent initiation of DNA synthesis that may be relevant to strand initiation events occurring during lambda DNA replication.

Bacteriophage lambda↗
PubMed2000

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↗
PubMed1992

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↗
PubMed1997

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↗
PubMed1995

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↗
PubMed1991

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↗
PubMed1995

Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication.

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.

DNA Helicases↗
PubMed1999

Bloom's syndrome protein, BLM, colocalizes with replication protein A in meiotic prophase nuclei of mammalian spermatocytes.

Bloom's syndrome (BS) is a rare autosomal recessive disorder of humans characterized by severe pre- and postnatal growth deficiency, immunodeficiency, genomic instability, and a predisposition to a wide variety of neoplasms. The genomic instability is evidenced in BS somatic cells as a high incidence of gaps and breaks, chromatid exchanges, chromosome rearrangements, and locus-specific mutations. BS arises from a mutation in BLM, a gene encoding a protein with homology to the RecQ helicase family. Men with BS are sterile; women have reduced fertility and a shortened reproductive span. The current immunocytological study on mouse spermatocytes shows that the BLM protein is first evident as discrete foci along the synaptonemal complexes (SCs) of homologously synapsed autosomal bivalents in late zygonema of meiotic prophase. BLM foci progressively dissociate from the synapsed autosomal axes during early pachynema and are no longer seen in mid-pachynema. BLM colocalizes with the single-stranded DNA binding replication protein A, which has been shown to be involved in meiotic synapsis. However, there is a temporal delay in the appearance of BLM protein along the SCs relative to replication protein A, suggesting that BLM is required for a late step in processing of a subset of genomic DNA involved in establishment of interhomologue interactions in early meiotic prophase. In late pachynema and into diplonema, BLM is more dispersed in the nucleoplasm, especially over the chromatin most intimately associated with the SCs, suggesting a possible involvement of BLM in resolution of interlocks in preparation for homologous chromosome disjunction during anaphase I.

Adenosine Triphosphatases↗