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PubMed1994

Human replication protein A binds single-stranded DNA in two distinct complexes.

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

Base Sequence↗
PubMed1990

Localization of adenovirus-encoded DNA replication proteins in the nucleus by immunogold electron microscopy.

The distribution of three adenovirus-encoded DNA replication proteins in the nucleus of human 293 cells was studied by immunogold electron microscopy. The infected nuclei contained four morphologically distinct inclusions. They were highly electron-dense granules (type I), compact fibrogranular masses of medium electron density (type II), filamentous masses of low electron density (type III) and large polygonal crystals (type IV). In immunogold labelling studies, antibodies to the adenovirus single-stranded DNA-binding protein (DBP) and antibodies to single-stranded DNA showed extensive binding to the type III inclusions. The antibodies to the adenovirus DNA polymerase (AdPol) and terminal protein (TP) predominantly labelled type II inclusions. Double immunogold labelling studies detected low levels of AdPol and TP in type III inclusions and DBP in type II inclusions. The selective distribution of DNA replication proteins suggests that the type II and III inclusions represent two functionally different entities that may be involved in two different aspects of adenovirus DNA replication, i.e. chain initiation and elongation.

Adenoviridae↗
PubMed2004

Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity.

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.

DNA-Binding Proteins↗
PubMed1996

Dynamics of human replication protein A subunit distribution and partitioning in the cell cycle.

Human replication protein A (RPA) is a three-sub-unit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In meta-phase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.

Cell Cycle↗
PubMed2002

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.

Animals↗
PubMed1995

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.

Base Sequence↗
PubMed1998

Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro.

Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6-fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.

Cisplatin↗
PubMed2004

DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A.

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.

DNA Polymerase beta↗
PubMed1988

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins.

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.

Bacterial Proteins↗
PubMed1984

Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA.

The purified bacteriophage lambda replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of lambda O and one molecule of lambda P. The lambda O-P oligomer is active in the in vitro replication of ori lambda-containing DNA. Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the lambda replication proteins and ori lambda DNA. It was found that the lambda P protein binds specifically to ori lambda-containing plasmid DNA only in the presence of lambda O protein. About 100 molecules of lambda O and 10 molecules of lambda P form a complex with the ori lambda DNA. The lambda DNA-lambda O-lambda P complex was shown to be active in an in vitro replication system. Since the physical interactions between ori lambda and lambda O and between lambda P and the Escherichia coli dnaB replication protein are well documented, the evidence for a lambda O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables lambda to direct the host replication machinery to the replication of its own DNA.

Bacteriophage lambda↗
PubMed2005

Bacillus subtilis bacteriophage SPP1-encoded gene 34.1 product is a recombination-dependent DNA replication protein.

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.

Adenosine Triphosphate↗
PubMed1992

ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes.

The PriA replication protein of Escherichia coli (formerly replication factor Y or protein n') is multifunctional. It is a site-specific, single-stranded DNA-dependent ATPase (dATPase), a 3'----5' DNA helicase, and guides the ordered assembly of the primosome, a mobile, multiprotein DNA replication priming/helicase complex. Although PriA is not absolutely required for viability, priA null mutant cells grow very slowly, have poor viability, and form extensive filaments. In order to assess which of the multiple activities of PriA are required for normal replication and growth, site-directed mutagenesis was employed to introduce single amino acid substitutions for the invariant lysine within the consensus nucleotide-binding motif found in PriA. Biochemical characterization of the representative purified mutant PriA proteins revealed them to be completely deficient in nucleotide hydrolysis, incapable of translocation along a single-stranded DNA binding protein-coated single-stranded DNA template, and unable to manifest the 3'----5' DNA helicase activity of wild-type PriA. These mutant proteins were, however, capable of catalyzing the assembly of active primosomes in vitro. Furthermore, when supplied in trans to insertionally inactivated priA cells, plasmids containing a copy of these mutant priA genes restored the wild-type growth rate, viability, and cell morphology. Based on these results, a model for PriA function in vivo is discussed.

Adenosine Triphosphatases↗
PubMed2000

Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response.

The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.

Cell Cycle↗
PubMed2003

The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction.

Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.

Animals↗
PubMed1994

Isolation of the genes encoding the 51-kilodalton and 28-kilodalton subunits of Crithidia fasciculata replication protein A.

The genes encoding the 51-kilodalton subunit (p51) and the 28-kilodalton subunit (p28) of replication protein A (RP-A), designated CfaRPA1 and CfaRPA2 respectively, were cloned from the trypanosomatid Crithidia fasciculata by screening a genomic DNA library in the expression vector lambda gt11 with antibodies raised against purified C. fasciculata RP-A. CfaRPA1 has a single open reading frame encoding a polypeptide of 467 amino acids and a molecular mass of 52.0 kDa. The predicted p51 polypeptide has sequence similarity to the corresponding subunits from human, Xenopus laevis, and Saccharomyces cerevisiae, but is lacking a segment of approximately 20 kDa from its amino terminus, accounting for its smaller molecular weight when compared to the large subunits of RP-A from these other organisms. CfaRPA1 contains a zinc-finger motif that is also found in the RP-A large subunits from human, frog, and yeast. CfaRPA2 contains a single large open reading frame encoding a polypeptide of 258 amino acids and a molecular mass of 27.5 kDa. The predicted polypeptide has significant sequence similarity to the middle subunit of RP-A from human cells, mouse cells, and the budding yeast S. cerevisiae. Northern hybridization analysis of polyadenylated RNA from C. fasciculata indicates that both cloned genes are expressed as polyadenylated transcripts. CfaRPA1 hybridized with a 2.30-kb transcript and CfaRPA2 hybridized with a 1.44-kb transcript.

Amino Acid Sequence↗
PubMed2001

Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4.

DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein alpha. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in alpha (alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on lambda immunity in E.coli for in vivo detection of protein-protein interactions, we found that (i) alpha protein interacts with Cnr, whereas alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr) interactions occur and the interaction domain is located within the C-terminal of alpha; (iii) Cnr-Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both alpha-alpha and alpha(cr)-alpha(cr) dimerization. Our data suggest that Cnr and alpha interact in at least two ways, which may have different functional roles in P4 replication control.

Amino Acid Substitution↗
PubMedDate not supplied

[Interaction of human replication protein A with DNA-duplexes, containing gaps of varying sizes].

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3' end of a gap-flanking oligonucleotide with DNA polymerase beta, and an oligonucleotide containing a 5'-photoreactive group was chemically synthesized. The 5' end predominantly interacted with the large RPA subunit (p70) regardless of the gap size, whereas interactions of the 3' end with the RPA subunits depended both on the gap size and on the RPA concentration. Subunit p32 was mostly labeled in the case of a larger gap and a lower RPA concentration. The results confirmed the model of polar RPA-DNA interaction, which has been advanced earlier.

Base Sequence↗
PubMed1997

Phosphorylation of the adeno-associated virus replication proteins.

The adeno-associated virus (AAV) replication proteins Rep78 and Rep68 regulate viral gene expression and DNA amplification. Their effects on both processes suggest that they play roles in all phases of the virus life cycle. We have investigated Rep protein phosphorylation to determine if this modification might alter Rep function. All four Rep proteins were found to be phosphorylated in AAV and adenovirus co-infected cell cultures, and Rep proteins contained phospho-serine whereas no phospho-threonine or -tyrosine was detected. We also observed that when viral DNA synthesis was inhibited, there was a significant decrease in the level of Rep78/68 phosphorylation. Our results suggest a plausible mechanism whereby AAV Rep 78/68 function may be regulated by phosphorylation.

Cell Line, Transformed↗