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PubMed2006

Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes.

We have examined the single-stranded DNA (ssDNA) binding properties of the Saccharomyces cerevisiae replication protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants, and binding enthalpy of scRPA-(dT)L complexes as a function of the oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is a stable heterotrimer over the range of [NaCl] examined (0.02-1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n = 18-20 nucleotides at low [NaCl] and values of n = 26-28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0 degrees C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT)L complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in the occluded site size. Measurements of the deltaH(obsd) for scRPA binding to (dT)L at 1.5 M NaCl yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 +/- 1 nucleotides), stabilized at low [NaCl], in which only three of its oligonucleotide/oligosaccharide binding folds (OB-folds) are used, and a higher site size mode (n = 27 +/- 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found under any conditions examined. Thus, scRPA shows some behavior similar to that of the E. coli SSB homotetramer, which also shows binding mode transitions, but some significant differences also exist.

DNA, Single-Stranded↗
PubMed1999

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions.

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.

DNA↗
PubMed1994

Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Base Sequence↗
PubMed1991

The priB and priC replication proteins of Escherichia coli. Genes, DNA sequence, overexpression, and purification.

The Escherichia coli DNA replication proteins n and n" function in vitro in the assembly of the primosome, a mobile multiprotein replication priming complex thought to operate on the lagging-strand template at the E. coli DNA replication fork. Both proteins have been purified from E. coli HMS83 cells based on their requirement for the reconstitution of bacteriophage phi X174 complementary strand DNA synthesis in vitro with purified proteins. As a step toward understanding the role of these proteins in vivo, the genes for primosomal proteins n and n", designated priB and priC, respectively, have been cloned molecularly. priB encodes a 104-amino acid 11.4-kDa polypeptide and corresponds to an previously identified open reading frame between rpsF and rps R within a ribosomal protein operon at 95.5 min on the E. coli chromosome. priC encodes a 175-amino acid 20.3-kDa polypeptide. These two gene products were overexpressed at least 1000-fold in E. coli using a bacteriophage T7 transient expression system. Both proteins have been purified to apparent homogeneity from extracts prepared from these overproducing strains.

Amino Acid Sequence↗
PubMed2000

Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A.

We describe the unique structural features of a large telomere repeat DNA complex (TRDC) of >20 kb generated by a simple PCR using (TTAGGG)(4) and (CCCTAA)(4) as both primers and templates. Although large, as determined by conventional agarose gel electrophoresis, the TRDC was found to consist of short single-stranded DNA telomere repeat units of between several hundred and 3000 bases, indicating that it is a non-covalent complex comprising short cohesive telomere repeat units. S1 nuclease digestion showed that the TRDC contains both single- and double-stranded portions stable enough to survive glycerol density gradient centrifugation, precipitation with ethanol and gel electrophoresis. Sedimentation analysis suggests that a part of the TRDC is non-linear and consists of a three-dimensional network structure. After treatment with Werner DNA helicase the TRDC dissociated into smaller fragments, provided that human replication protein A was present, indicating that: (i) the TRDC is a new substrate for the Werner syndrome helicase; (ii) the telomere repeat sequence re-anneals rapidly unless unwound single-stranded regions are protected by replication protein A; (iii) the TRDC may provide a new clue to understanding deleterious telomere-totelomere interactions that can lead to genomic instability. Some properties of the TRDC account for the extra-chromosomal telomere repeat (ECTR) DNA that exists in telomerase-negative immortalized cell lines and may be involved in maintaining telomeres.

Centrifugation, Density Gradient↗
PubMed1995

Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range.

pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon.

Amino Acid Sequence↗
PubMed1992

Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.

3T3 Cells↗
PubMed1996

Presence of antibodies to replication protein A in some patients with systemic lupus erythematosus (SLE).

Presence of antibodies directed against replication protein A (RPA), a DNA binding protein complex composed of three subunits (RPA-70, RPA-32 and RPA-14) was investigated among patients with SLE and other autoimmune diseases using immunoblot analysis to RPA-70 and RPA-32 recombinant proteins. Anti-RPA antibodies were found in two out of 108 sera from SLE patients, one of them showing reactivity against RPA-32 and RPA-70 and the other reacting only against RPA-32. Sera from 108 patients with other autoimmune disorders as well as from 42 healthy control individuals were negative. Thus, the frequency of these antibodies in SLE is estimated to be 2-3%. The study demonstrates that RPA is one target more of the wide array of autoantigens that elicit an immune response in SLE. The presence of anti-RPA autoantibodies seems to be circumscribed to a small number of patients with SLE.

Adult↗
PubMed2005

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus.

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.

Bacterial Proteins↗
PubMed1989

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.

Antigens, Polyomavirus Transforming↗
PubMed2002

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A.

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.

Adenosine Triphosphate↗
PubMed2004

Replication protein A interacts with AID to promote deamination of somatic hypermutation targets.

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA deaminase required for somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes. Class switch recombination involves transcription through switch regions, which generates ssDNA within R loops. However, although transcription through immunoglobulin variable region exons is required for SHM, it does not generate stable ssDNA, which leaves the mechanism of AID targeting unresolved. Here we characterize the mechanism of AID targeting to in-vitro-transcribed substrates harbouring SHM motifs. We show that the targeting activity of AID is due to replication protein A (RPA), a ssDNA-binding protein involved in replication, recombination and repair. The 32-kDa subunit of RPA interacts specifically with AID from activated B cells in a manner that seems to be dependent on post-translational AID modification. Thus, our study implicates RPA as a novel factor involved in immunoglobulin diversification. We propose that B-cell-specific AID-RPA complexes preferentially bind to ssDNA of small transcription bubbles at SHM 'hotspots', leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins.

Amination↗
PubMed2004

Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities.

ATR is an essential protein that functions as a damage sensor and a proximal kinase in the DNA damage checkpoint response in mammalian cells. It is a member of the phosphoinositide 3-kinase-like kinase (PIKK) family, which includes ATM, ATR, and DNA-dependent protein kinase. Recently, it was found that ATM is an oligomeric protein that is converted to an active monomeric form by phosphorylation in trans upon DNA damage, and this raised the possibility that other members of the PIKK family may be regulated in a similar manner. Here we show that ATR is a monomeric protein associated with a smaller protein called ATRIP with moderate affinity. The ATR protein by itself or in the form of the ATR-ATRIP heterodimer binds to naked or replication protein A (RPA)-covered DNAs with comparable affinities. However, the phosphorylation of RPA by ATR is dependent on single-stranded DNA and is stimulated by ATRIP. These findings suggest that the regulation and mechanism of action of ATR are fundamentally different from those of the other PIKK proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Binding activity of replication protein A to UV-damaged single-stranded DNA.

To obtain the information for the exact role of replication protein A (RPA) on both eukaryotic DNA replication and repair, the binding preference of RPA purified from Xenopus egg extract against the undamaged and UV-damaged single-stranded DNA was studied by the gel shift assay. Chemically synthesized oligonucleotide containing the pyrimidine(6-4)pyrimidone photoproduct at one site was used as a model of UV-damaged DNA. Results of competition assay and Scatchard plots indicate that RPA preferentially binds to the 6-4 photoproduct oligonucleotide than the undamaged DNA.

Animals↗
PubMed2004

The interaction of p53 with replication protein A mediates suppression of homologous recombination.

The tumor suppressor protein p53 is emerging as a central regulator of homologous recombination (HR) processes and DNA replication. P53 may downregulate HR through multiple mechanisms including the reported associations with the Rad51 and Rad54 recombinases, and the BLM and WRN helicases. Here, we investigated whether the interaction of p53 with human replication protein A (RPA) is necessary for the regulation of HR. By employing a plasmid-based HR assay in p53-null H1299 lung carcinoma cells, we studied the HR-suppressing properties of a panel of p53 mutants, which varied in their ability to interact with RPA. Both wild-type p53 and a transactivation-deficient p53 mutant (L22Q/W23S) suppressed HR and prevented RPA binding to ssDNA in vitro and in vivo. Conversely, p53 mutations that specifically disrupt the RPA-binding domain, while not compromising p53 transactivation function (D48H/D49H and W53S/F54S), did not affect HR. Suppression of HR was also not seen with missense mutations in the p53 core domain (His175 and His273), which retained the ability to interact with RPA, suggesting that the disruption of additional binding interactions of p53, for example, with Rad51 or recombination intermediates, also impacts on HR. We hypothesize that sequestration of RPA by p53 at the sites of recombination is one means by which p53 can inhibit HR processes. Our data support and extend the previously formulated 'dual model' of p53's role as guardian of the genome.

Base Sequence↗