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PubMed1997

Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase.

The RAD51 and RAD52 genes of Saccharomyces cerevisiae are key members of the RAD52 epistasis group required for genetic recombination and the repair of DNA double-stranded breaks. The RAD51 encoded product mediates the DNA strand exchange reaction. Efficient strand exchange is contingent upon the addition of the heterotrimeric single-stranded DNA binding factor replication protein A (RPA) after Rad51 has nucleated onto the single-stranded DNA. However, if the single-stranded DNA is incubated with Rad51 and RPA simultaneously to mimic what may be expected to occur in vivo, the efficiency of strand exchange decreases dramatically, revealing an inhibitory effect of RPA that is distinct from its stimulatory function. Interestingly, the inclusion of Rad52 protein, which has been purified in this study from yeast cells, restores the efficiency of strand exchange. Thus, Rad52 functions as a co-factor for the Rad51 recombinase, acting specifically to overcome the apparent competition by RPA for binding to single-stranded DNA.

DNA Helicases↗
PubMed2006

Functional assays for replication protein A (RPA).

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein. RPA is conserved in all eukaryotes and is essential for DNA replication, DNA repair, and recombination. RPA also plays a role in coordinating DNA metabolism and the cellular response to DNA damage. Assays have been established for many of these reactions. This chapter provides an overview of the methods used for analyzing RPA-DNA interactions, RPA-protein interactions, and functional activities of RPA. Methods are also discussed for visualizing RPA in the cell and analyzing the effects of RPA function on cell cycle progression in mammalian cells.

Chromatography, Affinity↗
PubMed1992

Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins.

It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family. Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA. These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication. It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick. The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain. By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately. The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed.

Amino Acid Sequence↗
PubMed1993

The adenovirus terminal protein influences binding of replication proteins and changes the origin structure.

The adenovirus terminal protein (TP) is covalently linked to the 5' ends of the adenovirus genome and enhances DNA replication in vitro by increasing template activity. To study the effect of TP in more detail we isolated short origin fragments containing functional TP using anion exchange chromatography. These fragments were highly active as templates for DNA replication in a reconstituted system. Employing band-shift assays we found that the affinity of the precursor terminal protein-DNA polymerase complex for the TP-containing origin was increased 2 to 3-fold. Binding affinities of two other replication stimulating proteins, NFI and Oct-1, were not influenced by the terminal protein. Upon DNaseI footprinting we observed, unexpectedly, that the breakdown pattern had changed at various positions in the origin, notably in the area 3-6 and 41-51 by the presence of TP. Some differences in the footprint pattern of NFI and Oct-1 were also found. Our results indicate that TP induces subtle changes in the origin structure that influence the interaction of other replication proteins.

Adenoviridae↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed2005

Sumoylation of the novel protein hRIP{beta} is involved in replication protein A deposition in PML nuclear bodies.

Replication protein A (RPA) is a single-stranded-DNA-binding protein composed of three subunits with molecular masses of 70, 32, and 14 kDa. The protein is involved in multiple processes of eukaryotic DNA metabolism, including DNA replication, repair, and recombination. In Xenopus, Xenopus RPA-interacting protein alpha has been identified as a carrier molecule of RPA into the nucleus. In this study, human RPA-interacting protein alpha (hRIPalpha) and five novel splice isoforms (named hRIPalpha, hRIPbeta, hRIPgamma, hRIPdelta1, hRIPdelta2, and hRIPdelta3 according to the lengths of their encoding peptides) were cloned. Among hRIP isoforms, hRIPalpha and hRIPbeta were found to be the major splice isoforms and to show different subcellular localizations. While hRIPalpha localized to the cytoplasm, hRIPbeta was found in the PML nuclear body. Modification of hRIPbeta by sumoylation was found to be required for localization to the PML nuclear body. The results of the present work demonstrate that hRIPbeta transports RPA into the PML nuclear body and releases RPA upon UV irradiation. hRIPbeta thus plays an important role in RPA deposition in PML nuclear bodies and thereby supplements RPA for DNA metabolism.

Active Transport, Cell Nucleus↗
PubMed2002

Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum.

Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism. Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain. Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C. parvum genome-sequencing project. The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C. parvum. The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein. However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum. Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites. However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites. Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays. The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties. Studies indicate that CpRPA1B binds ssDNA of >or=5 nucleotides (dT), while CpRPA1 only binds ssDNA >or=20 nucleotides (dT). This study indicates that C. parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts.

Amino Acid Sequence↗
PubMed1995

Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein.

The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.

Amino Acid Sequence↗
PubMed2006

The dispersal of replication proteins after Etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway.

In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended single-stranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposide-induced inhibition of DNA replication in early S phase.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Stability and nuclear distribution of mammalian replication protein A heterotrimeric complex.

Replication protein A (RPA), a stable complex of three polypeptides, is the single-stranded DNA-binding protein essential for DNA replication in eukaryotic cells. Previous studies of the subcellular distribution and stability of the RPA heterotrimer during the mammalian cell cycle have produced conflicting results. Here, we present evidence that these inconsistencies can be accounted for by the presence of an extractable pool of soluble RPA within the nucleus. Indirect immunofluorescence experiments in both CHO and HeLa cells showed that all three RPA subunits associated specifically with sites of ongoing DNA synthesis, similar to the replication fork protein proliferating cell nuclear antigen. Furthermore, we found no evidence for disassembly of the chromatin-bound heterotrimeric RPA complex in vivo. Our results are consistent with a role for RPA in the initiation and elongation steps of replication, as previously defined in the viral in vitro replication systems.

Animals↗
PubMed1995

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.

Base Sequence↗
PubMed2002

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen.

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.

Animals↗
PubMed1998

The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair.

The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases alpha and delta, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1-278 or 222-411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair.

Binding Sites↗
PubMed1997

Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase alpha by multiple mechanisms.

Replication Protein A (RPA) from human cells is a stable complex of 70-, 32-, and 14-kDa subunits that is required for multiple processes in DNA metabolism. RPA binds with high affinity to single-stranded DNA and interacts with multiple proteins, including proteins required for the initiation of SV40 DNA replication, DNA polymerase alpha and SV40 large T antigen. We have used a series of mutant derivatives of RPA to map the regions of RPA required for specific protein-protein interactions and have examined the roles of these interactions in DNA replication. T antigen, DNA polymerase alpha and the activation domain of VP16 all have overlapping sites of interaction in the N-terminal half (residues 1-327) of the 70-kDa subunit of RPA. In addition, the interaction site for DNA polymerase alpha is composed of two functionally distinct regions, one (residues 1- approximately 170) which stimulates polymerase activity and a second (residues approximately 170-327) which increases polymerase processivity. In the latter, both the direct protein-protein interaction and ssDNA-binding activities of RPA were needed for RPA to modulate polymerase processivity. We also found that SV40 T antigen inhibited the ability of RPA to increase processivity of DNA polymerase alpha, suggesting that this activity of RPA may be important for elongation but not during the initiation of DNA replication. DNA polymerase alpha, but not T antigen also interacted with the 32- and/or 14-kDa subunits of RPA, but these interactions did not seem to effect polymerase activity.

Antigens, Polyomavirus Transforming↗
PubMed2005

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair.

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.

Cisplatin↗
PubMed2004

A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein.

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted alpha-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.

Amino Acid Motifs↗
PubMed2003

Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein.

Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs. We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Delta9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs). Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis. We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane. 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from approximately 2.5 to approximately 1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation. Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules. However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds. Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs. This localized UFA depletion helps to explain why BMV RNA replication is more sensitive than cell growth to reduced UFA levels. The results imply that 1a preferentially interacts with one or more types of membrane lipids.

Bromovirus↗
PubMed2000

A hypothesis for DNA viruses as the origin of eukaryotic replication proteins.

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.

Amino Acid Sequence↗