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PubMed1994

Interactions of human replication protein A with oligonucleotides.

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding protein that is essential for eukaryotic DNA replication. In order to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stranded oligonucleotides. Our analysis of RPA.DNA complexes demonstrated that RPA binds as a heterotrimer. Stoichiometric binding reactions monitored by fluorescence quenching indicated that the binding site size of human RPA is 30 nucleotides and that between 20-30 nucleotides of DNA directly interact with RPA. The binding of RPA to DNA of different lengths was systematically examined using deoxythymidine-containing oligonucleotides. We found that the binding affinity of RPA for short oligonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from approximately 7 x 10(7) M-1 for oligo(dT)10 to approximately 1.5 x 10(10) M-1 for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (omega) for RPA binding was estimated to be approximately 15.

Adenosine Triphosphate↗
PubMed1997

Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase.

Saccharomyces cerevisiae RAD51, RAD55, and RAD57 genes, required for genetic recombination and DNA double-strand-break repair, encode proteins homologous to one another and to the Escherichia coli RecA protein. Rad51 protein catalyzes the DNA strand-exchange reaction with a dependence on ATP and on the heterotrimeric single-strand DNA (ssDNA) binding factor replication protein A (RPA). By several independent criteria, RAD55- and RAD57-encoded products are shown here to exist as a stable heterodimer, with a dissociation constant of <2 x 10(-10) M. In strand exchange, the reaction proceeds efficiently if RPA is incorporated after nucleation of Rad51 onto ssDNA, but if RPA is present during the nucleation phase, as is likely the case in vivo, the amount of strand-exchange products becomes relatively insignificant. Inclusion of the Rad55-Rad57 heterodimer with Rad51 and RPA results in a marked stimulation of strand exchange, providing evidence for a role of the Rad55-Rad57 heterodimer in overcoming the inhibitory effect of RPA.

Adenosine Triphosphatases↗
PubMed1999

Roles of replication protein A and DNA-dependent protein kinase in the regulation of DNA replication following DNA damage.

Exposure of mammalian cells to DNA damage-inducing agents (DDIA) inhibits ongoing DNA replication. The molecular mechanism of this inhibition remains to be elucidated. We employed a simian virus 40 (SV40) based in vitro DNA replication assay to study biochemical aspects of this inhibition. We report here that the reduced DNA replication activity in extracts of DDIA-treated cells is partly caused by a reduction in the amount of replication protein A (RPA). We also report that the dominant inhibitory effect is caused by the DNA-dependent protein kinase (DNA-PK) which inactivates SV40 T antigen (TAg) by phosphorylation. The results demonstrate that RPA and DNA-PK are involved in the regulation of viral DNA replication after DNA damage and suggest that analogous processes regulate cellular DNA replication with the DNA-PK targeting the functional homologues of TAg.

Androstadienes↗
PubMed1999

Functional studies on the interaction between human replication protein A and Xeroderma pigmentosum group A complementing protein (XPA).

The human replication protein A (RPA; also known as human single-stranded DNA binding protein, HSSB) is a multisubunit complex (70, 34 and 11 kDa subunits) involved in the three processes of DNA metabolism; replication, repair, recombination. We found that both 34 and 70 kDa subunits (p34 and p70, respectively), of RPA interacts with the Xeroderma pigmentosum group A complementing protein (XPA), a protein that specifically recognizes UV-damaged DNA. Our mutational analysis indicated that no particular domains of RPA p70 were essential for its interaction with XPA. We also examined the effect of this XPA-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the crude extract and monopolymerase system. XPA inhibited SV40 DNA replication in vitro through its interaction with RPA. Taken together, these results suggest that there is a role for RPA in the regulation of DNA metabolism through its ability to modulate the interactions of proteins involved in the processes of DNA metabolism.

Animals↗
PubMed1990

Host virus interactions in the initiation of bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB helicase by lambda P replication protein.

The bacteriophage lambda P protein promoters replication of the phage chromosome by recruiting a key component of the cellular replication machinery to the viral origin. Specifically, P protein delivers one or more molecules of Escherichia coli DnaB helicase to a nucleoprotein structure formed by the lambda O initiator at the lambda replication origin. Using purified proteins, we have examined the features of the pivotal host virus interaction between P and DnaB. These two proteins interact in vitro to form a P.DnaB protein complex that can be resolved by sedimentation or by chromatography on DEAE-cellulose from the individual free proteins. The sedimentation coefficient of the P.DnaB complex, 13 S, suggests a size larger than that of free DnaB hexamer (Mr = 313,600). The P.DnaB complex isolated by glycerol gradient sedimentation contains approximately three protomers of P/DnaB hexamer, consistent with a molecular weight of 393,000. The isolated P.DnaB complex functions in vitro in the initiation of lambda DNA replication. Interaction of P with DnaB strongly suppressed both the intrinsic DNA-dependent ATPase activity of DnaB, as well as the capacity of DnaB to assist E. coli primase in the general priming reaction. Formation of a P.DnaB protein complex also blocked DnaB from functioning in the initiation of E. coli DNA replication in vitro. The physical and functional properties of lambda P protein suggest that it is a viral analogue of the E. coli DnaC replication protein. Like P, DnaC also binds to DnaB (Wickner, S., and Hurwitz, J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925), but unlike P, DnaC stimulates DnaB-mediated general priming. When viral P and bacterial DnaC replication proteins were placed in direct competition with one another for binding to DnaB, the viral protein was clearly predominant. For example, a 5-fold molar excess of DnaC protein only partially reversed the inhibitory effect of P on general priming. Furthermore, when a preformed DnaC.DnaB protein complex was incubated briefly with P protein, it was readily converted into a P.DnaB protein complex and the bulk of the bound DnaC was released as free protein. It is likely that the capacity of the lambda P protein to outcompete the analogous host protein for binding to the bacterial DnaB helicase is the critical molecular event enabling infecting phage to recruit cellular replication proteins required for initiation of DNA synthesis at the viral origin.

Bacterial Proteins↗
PubMed1996

Replication protein A is a component of a complex that binds the human metallothionein IIA gene transcription start site.

Previous studies revealed that sequences surrounding the initiation sites in many mammalian and viral gene promoters, called initiator (Inr) elements, may be essential for promoter strength and for determining the actual transcription start sites. DNA sequences in the vicinity of the human metallothionein IIA (hMTIIA) gene transcription start site share homology with some of the previously identified Inr elements. However, in the present study we have found by in vitro transcription assays that the hMTIIA promoter does not contain a typical Inr. Electrophoretic mobility shift assays identified several DNA-protein complexes at the hMTIIA gene transcription start site. A partially purified protein fraction containing replication protein A (RPA) binds to the hMTIIA gene transcription start site and represses transcription from the hMTIIA promoter in vitro. In addition, overexpression of the human 70-kDa RPA-1 protein represses transcription of a reporter gene controlled by the hMTIIA promoter in vivo. These findings suggest that hMTIIA transcription initiation is controlled by a mechanism different from most mammalian and viral promoters and that the previously identified RPA may also be involved in transcription regulation.

Amino Acid Sequence↗
PubMed2005

Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication.

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase C. The related p28 replication protein of Turnip crinkle virus was also found to be phosphorylated in vivo, suggesting that posttranslational modification of replication proteins is a general feature among members of the large Tombusviridae family. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Phosphorylation-mimicking aspartic acid mutations rendered p33 nonfunctional in plant protoplasts and in yeast, a model host. Comparable mutations within the prereadthrough portion of p92 did not abolish replication. The nonphosphorylation-mimicking alanine mutants of CNV were able to replicate in plant protoplasts and in yeast, albeit with reduced efficiency when compared to the wild type. These alanine mutants also showed altered subgenomic RNA synthesis and a reduction in the ratio between plus- and minus-strand RNAs produced during CNV infection. These findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

Amino Acid Sequence↗
PubMed1982

Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.

Bacteriophage lambda↗
PubMed1994

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.

Base Sequence↗
PubMed1995

Recombinant human replication protein A binds to polynucleotides with low cooperativity.

Replication protein A (RPA) is a multisubunit single-stranded DNA-binding protein that is involved in multiple aspects of cellular DNA metabolism. We have determined quantitative estimates of the binding parameters of human replication protein A (hRPA) from equilibrium binding isotherms. The intrinsic binding constant (K) and cooperativity parameter (omega) were determined from analysis of changes in the intrinsic fluorescence of hRPA that occurred upon binding single-stranded DNA homopolynucleotides. The cooperativity of hRPA binding to both poly(dT) and poly(dA) was found to be low (omega = 10-20) at all NaCl concentrations examined (0.3-2 M). In contrast, the apparent binding affinity (K omega) of RPA decreased significantly with increasing salt concentration, such that log [NaCl]/log K omega was -2.8 for poly(dT) and -4.8 for poly(dA). We conclude that the salt dependent decrease in binding affinity resulted from changes in the intrinsic binding constant (K). These data suggest that the interaction of hRPA with single-stranded DNA involves significant electrostatic interactions, similar to other single-stranded DNA-binding proteins. The apparent binding affinity (K omega) of RPA was higher for poly(dT) than for poly(dA); extrapolation of our data indicated that the apparent binding affinity at 0.2 M NaCl was 1.6 x 10(10) M-1 for poly(dT) and 1.1 x 10(9) M-1 for poly(dA).

DNA, Single-Stranded↗
PubMed2001

Polarity of human replication protein A binding to DNA.

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5'-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5'-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3'-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.

Azides↗
PubMed2000

Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication.

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin-hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin-hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.

Cell Compartmentation↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1998

Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A.

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

Adenine Nucleotides↗
PubMed2001

Replication protein A as a "fidelity clamp" for DNA polymerase alpha.

The current view of DNA replication in eukaryotes predicts that DNA polymerase alpha (pol alpha)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol alpha elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol alpha displays a low processivity and, because of the lack of an intrinsic or associated 3'--> 5' exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol alpha-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol alpha playing a dual role: (i) it stabilizes the pol alpha/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol alpha. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol alpha.

Animals↗
PubMed2006

Thermodynamic analysis of the single-stranded DNA binding activity of the archaeal replication protein A (RPA) from Sulfolobus solfataricus.

The single-stranded DNA binding protein from Sulfolobus solfataricus (Sso-RPA) binds single-stranded DNA with dissociation constants in the range of 10-30 nM at room temperature. The affinity for DNA decreases at higher temperatures. At 85 degrees C, the optimal growth temperature of the crenarchaeot S. solfataricus, the dissociation constant is only about 1 microM. We analyzed the equilibrium between Sso-RPA and a fluorescently labeled 13 nucleotide oligonucleotide by fluorescence anisotropy measurements in the presence of four different salts and in the temperature range between 10 and 60 degrees C. In the presence of potassium chloride and choline chloride, three to four ions are released upon complexation, independent of the temperature. In contrast, in the presence of potassium fluoride and potassium glutamate, we observed a significant change of the number of ions released when the temperature was varied. The binding reaction is strongly exothermic with enthalpies of about -55 to -70 kJ/mol, depending upon the salt. Van't Hoff analysis suggests that the binding enthalpy is temperature independent.

Archaeal Proteins↗
PubMed1996

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

Base Sequence↗