Subunit Of Replication Protein A Research Articles

Mutations in the Gene Encoding the 34 kDa Subunit of Yeast Replication Protein A Cause Defective S Phase Progression

The in vivofunction of the 34 kDa subunit of yeast replication protein A (RPA), encoded by the RFA2gene, has been studied by analyzing the effect of Rpa34 depletion and by producing and characterizing rfa2temperature- sensitive mutants. We show that unbalanced stoichiometry of the RPA subunits does not affect cell growth and cell cycle progression until the level of Rpa34 becomes rate-limiting, at which point cells arrest with a late S/G2 DNA content. Rpa34 is involved in DNA replication in vivo, since rfa2ts mutants are defective in S phase progression and ARS plasmid stability, and rfa2 pol1double mutants are non-viable. Moreover, when shifted to the restrictive temperature, about 50% of the rfa2mutant cells rapidly die while traversing the S phase and the surviving cells arrest in late S/G2 at the RAD9checkpoint. Finally, rfa2mutant cells have a mutator and hyper-recombination phenotype and are more sensitive to hydroxyurea and methyl-methane-sulfonate than wild-type cells.

An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.

Rpa4, a Homolog of the 34-Kilodalton Subunit of the Replication Protein A Complex

Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-kDa subunit of RPA. Rpa4 associates with the 70- and 13-kDa subunits to form a trimeric complex capable of binding to single-stranded DNA. Rpa4 is preferentially expressed in placental and colon mucosa tissues. In the placenta, Rpa4 is more abundant than the 70-kDa Rpa1 subunit and is not associated with either Rpa1 or with any other single-stranded DNA-binding protein. In proliferating cells in culture, Rpa4 is considerably less abundant than Rpa1 and Rpa2. Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family.

The Role of the 34-kDa Subunit of Human Replication Protein A in Simian Virus 40 DNA Replication in Vitro

Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin-dependent kinase (Cdk) and DNA-dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C-terminally deleted p34 was only marginally active. This indicates that the C-terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase alpha, and bind to single-stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding.

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.

Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication.

Replication Protein A (RPA) is a multisubunit, single-stranded DNA-binding protein essential for DNA metabolism in eukaryotic cells. The 32-kDa subunit of RPA is phosphorylated in a cell cycle-dependent manner becoming phosphorylated during S phase. It has been postulated that this phosphorylation may regulate the activities of RPA and that the family of p34cdc2 kinases directly catalyzes the phosphorylation of RPA in the cell. We have mutated the two consensus p34cdc2 sites in the 32-kDa subunit of RPA individually and in combination and purified the mutant protein complexes. Mutant RPA with both consensus p34cdc2 sites converted to alanine was not phosphorylated by purified p34cdc2 kinase. Nevertheless, we found that the properties of these RPA mutants were identical to those of the wild-type protein. The mutated RPA proteins had normal single-stranded DNA binding activity and were completely functional for DNA replication. In addition, the mutants became hyperphosphorylated when incubated under DNA replication conditions. These results demonstrate that phosphorylation by p34cdc2 kinase is not essential for RPA function in DNA replication in vitro. Possible roles of RPA phosphorylation on DNA metabolism are discussed.

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.

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.

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.

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.

DNA unwinding by replication protein A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit.

Replication protein A (RP-A) is a heterotrimeric single-stranded DNA binding protein with important functions in DNA replication, DNA repair and DNA recombination. We have found that RP-A from calf thymus can unwind DNA in the absence of ATP and MgCl2, two essential cofactors for bona fide DNA helicases (Georgaki, A., Strack, B., Podust, V. and Hübscher, U. FEBS Lett. 308, 240-244, 1992). DNA unwinding by RP-A was found to be sensitive to MgCl2, ATP, heating and freezing/thawing. Escherichia coli single stranded DNA binding protein at concentrations that coat the single stranded regions had no influence on DNA unwinding by RP-A suggesting that RP-A binds fast and tightly to single-stranded DNA. DNA unwinding by RP-A did not show directionality. Experiments with monoclonal antibodies strongly suggested that the 70kDa subunit is responsible for DNA unwinding. Phosphorylation of the 32kDa subunit of RP-A by chicken cdc2 kinase facilitated DNA unwinding indicating that this posttranslational modification might be important for modulating this activity of RP-A. Finally, DNA unwinding of a primer recognition complex for DNA polymerase delta which is composed of proliferating cell nuclear antigen, replication factor C and ATP bound to a singly-primed M13DNA slightly inhibited DNA unwinding. An important role for DNA unwinding by RP-A in processes such as initiation of DNA replication, fork propagation, DNA repair and DNA recombination is discussed.

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.

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.

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.