Polymerase Alpha Complex Research Articles

Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival.

Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion. The essential function is believed to result from its role as a Pol1p (polymerase alpha) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p. In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage. Characterization of an inducible allele of mcl1+, (nmt41)mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Delta, rqh1Delta, and hsk1-1312. Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea. We take these data to mean that Mcl1p is a dynamic component of the polymerase alpha complex during replication and is important for the replication stress response in fission yeast.

Purification and characterization of DNA polymerase alpha-associated replication protein A-dependent yeast DNA helicase A.

A novel, eukaryotic, hexameric DNA helicase that was earlier identified as a component of the multiprotein polymerase alpha complex [Biswas et al. (1993) Biochemistry 32, 13393-13398] has been purified to homogeneity and characterized. Thus far, our studies demonstrated that helicase A shares certain unique features of two other hexameric DNA helicases: the DnaB helicase of Escherichia coli and the T-antigen helicase of the SV40 virus. The helicase activity was stimulated by yeast replication protein A (RPA) and to a lower extent by E. coli single-stranded DNA binding protein (SSB). The helicase had an apparent molecular mass of 90 kDa, as determined by its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A tryptic peptide fragment of the polypeptide was sequenced followed by a BLAST search of GenBank with the tryptic peptide sequence. The search identified a 1.8 kb open reading frame previously designated as ykl017c on chromosome XI, that codes for a 78.3 kDa (683 amino acid) polypeptide. The important features of the polypeptide sequence of helicase A included a type I ATP/GTP binding motif, and a K E E R R L N V A M T R P R R sequence at the C-terminus that may be indicative of a nuclear localization signal which is required of a nuclear DNA helicase. The polypeptide sequence of helicase A appears to have homology to the DnaB helicase of E. coli (approximately 25%). The facts that these two helicases are vastly separated by evolution and retained similar structural and functional features, as demonstrated here, point to a possible significance of this limited homology. Although the amount of purified helicase A was limited, we have carried out necessary enzymatic characterization so that these data could be correlated with that of immunoaffinity-purified helicase A and recombinant helicase A expressed in heterologous systems.

Purification and characterization of a yeast DNA polymerase alpha complex with associated primase, 5'-->3' exonuclease, and DNA-dependent ATPase activities.

We have purified a multimeric form of yeast DNA polymerase alpha with DNA polymerase, primase, 5'-->3' exonuclease, and single-stranded (ss) DNA-dependent ATPase activities to near-homogeneity. The molecular mass of complex was 650 kDa with subunits ranging in sizes from 30 to 180 kDa. The alpha-subunit of the complex could be detected by DNA polymerase alpha antibody. No cross-reactivity of polypeptides within the complex was observed with antibodies directed against polymerase delta or epsilon. The multimeric polymerase alpha could be selectively inhibited by p-n-butylphenyl-dGTP (I50 of approximately 0.2 microM), p-n-butylanilino-dATP (I50 of 1.3 microM), and aphidicolin (I50 of 2.5 micrograms/mL). The complex synthesized RNA primers on various ssDNA templates and rapidly elongated these primers into nascent DNA fragments in the presence of required deoxynucleotides. It has a strong 5'-->3' exonuclease activity. In addition, the complex hydrolyzed both ATP and dATP in a ssDNA-dependent manner. Thus, the multiprotein complex of DNA polymerase alpha had multiple activities (primase, polymerase, and ATPase) which could act concertedly to synthesize primers and elongate the primers to nascent DNA fragments in the lagging strand of the fork.

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase alpha complex.

We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of simian virus 40 large T-antigen with cellular DNA polymerase alpha: studies with various T-antigen mutants of SV40.

Simian virus 40 (SV40) large T antigen binds to host cell DNA polymerase alpha and the T-antigen-DNA polymerase alpha complex is implicated in the initiation of viral DNA replication. We have examined various SV40 T-antigen mutants to test the correspondence between viral DNA replication and T-antigen-DNA polymerase alpha complex formation. The various SV40 T-antigen mutants were used to either infect or transfect African green monkey kidney cell line CV-1, and at different time intervals we measured the production of T-antigen and host cell DNA polymerase alpha by radioimmunoassay, complex formation by a sandwich radioimmunoassay and the amount of viral DNA synthesis by dot-blot hybridization analysis. There was a good correlation between complex formation and viral DNA synthesis in lytic mutants of SV40. Poor complex formation and correspondingly lower DNA synthesis were observed in the non-viable mutants of SV40, even though significant amounts of T-antigen and DNA polymerase alpha were present. Our results substantiate the earlier findings of T-antigen-DNA polymerase alpha complex formation and establish the need for formation of this complex in promoting viral DNA synthesis.

Single-stranded DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase .alpha.-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.

DNA polymerase‐primase complex in wild‐type and ts A1S9 mouse L‐cells, temperature‐sensitive for dna replication during cell cycle progression

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.

DNA primase stimulatory factor from mouse FM3A cells has an RNase H activity. Purification of the factor and analysis of the stimulation.

Two forms of DNA primase stimulatory factor have been purified from mouse FM3A cells and shown to have RNase H activity. One of the factors, which consists of three polypeptides of 42,000, 41,000, and 27,000 daltons, was characterized in its properties as RNase H and DNA primase stimulatory factor. The nucleolytic activity of the factor specifically digested the RNA component of RNA-DNA hybrids in an endonucleolytic manner. The stimulation by the factor was observed in DNA synthesis by DNA primase-DNA polymerase alpha complex on unprimed DNA templates, and the DNA chains synthesized under these conditions in the presence of the factor were much shorter than those synthesized in its absence. The stimulatory effect of the factor on DNA primase activity was directly confirmed with DNA primase dissociated from DNA polymerase alpha by the observation of the increase in the number of synthesized oligoribonucleotides. The primer RNA synthesis by DNA primase-DNA polymerase alpha complex under the condition where DNA synthesis occurred was also significantly stimulated by the factor. Furthermore, under these conditions RNA primers were removed from DNA chains by the RNase H activity of the factor.

Purification and characterization of primer recognition proteins from HeLa cells

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.

DNA polymerase α from normal rat liver is different than DNA polymerases α from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.

DNA primase-DNA polymerase α assembly from mouse FM3A cells: Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis

The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.

Phosphorylation of HeLa Cell Multiprotein DNA Polymerase α Complex: Impact on Activity and Partial Purification of the Associated Kinase

Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site.

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.

Affinity labeling the DNA polymerase alpha complex. I. Pyridoxal 5′-phosphate inhibition of DNA polymerase and DNA primase activities of the DNA polymerase alpha complex from Drosophila melanogaster embryos.

DNA polymerase alpha from Drosophila melanogaster embryos is a multisubunit enzyme complex which can exhibit DNA polymerase, 3'----5' exonuclease, and DNA primase activities. Pyridoxal 5'-phosphate (PLP) inhibition of DNA polymerase activity in this complex is time dependent and exhibits saturation kinetics. Inhibition can be reversed by incubation with an excess of a primary amine unless the PLP-enzyme conjugate is first reduced with NaBH4. These results indicate that PLP inhibition occurs via imine formation at a specific site(s) on the enzyme. Results from substrate protection experiments are most consistent with inhibition of DNA polymerase activity by PLP binding to either one of two sites. One site (PLP site 1) can be protected from PLP inhibition by any nucleoside triphosphate in the absence or presence of template-primer, suggesting that PLP site 1 defines a nucleotide-binding site which is important for DNA polymerase activity but which is distinct from the DNA polymerase active site. PLP also inhibits DNA primase activity of the DNA polymerase alpha complex, and primase activity can be protected from PLP inhibition by nucleotide alone, arguing that PLP site 1 lies within the DNA primase active site. The second inhibitory PLP-binding site (PLP site 2) is only protected from PLP inhibition when the enzyme is bound to both template-primer and correct dNTP in a stable ternary complex. Since binding of PLP at site 2 is mutually exclusive with template-directed dNTP binding at the DNA polymerase active site, PLP site 2 appears to define the dNTP binding domain of the active site. Results from initial velocity analysis of PLP inhibition argue that there is a rate-limiting step in the polymerization cycle during product release and/or translocation.

Identification and subcellular localization of the polypeptide for chick DNA primase with a specific monoclonal antibody.

Polypeptides responsible for activities of chick embryo DNA primase and DNA polymerase alpha were identified using monoclonal antibodies specific to these two enzymes. The 4-8H antibody neutralized DNA polymerase alpha activity measured on activated DNA template and also ribonucleoside triphosphate-dependent DNA synthesis on single-stranded DNA template (DNA primase-DNA polymerase alpha combined activity) to a partial extent (about 30%), but did not affect DNA primase activity. The 4-2D antibody, although it did not affect DNA polymerase alpha activity, did neutralize both DNA primase activity and DNA primase-DNA polymerase alpha combined activity extensively (up to 70%). Immunoblotting analysis of the DNA primase-DNA polymerase alpha complex showed that 4-2D and 4-8H antibodies recognize 60-kDa and 160-180-kDa polypeptides, respectively. An immunoaffinity column made of either of these antibodies retained DNA primase-DNA polymerase alpha complex. When the enzyme was eluted from the 4-8H column with alkaline solution, DNA primase was eluted prior to DNA polymerase alpha. In the case of 4-2D antibody column chromatography, the elution order of two enzymes was reversed. Results indicate that two enzymes in the complex which was retained in the antibody column were dissociated by lower alkaline pH than that dissociated the antigenic enzymes from the corresponding antibodies. In both cases, the fractions with DNA primase activity contained exclusively 60-kDa polypeptide, while those with DNA polymerase alpha contained 160-180-kDa polypeptides. Thus, DNA primase resided in 60-kDa polypeptide and was recognized by 4-2D antibody while DNA polymerase alpha resided in 160-180-kDa polypeptides and was recognized by 4-8H antibody. Immunofluorescence made with the DNA primase-specific 4-2D antibody as well as with 4-8H antibody appeared in granular structures which were tightly bound to the nuclear matrix. These nuclear fluorescences were much reduced in quiescent cells. Furthermore, since the fluorescence made by these antibodies was induced by adding serum to the quiescent cells in serum-deprived cultures, the expression of DNA primase and its organization in the structures on the nuclear matrix are regulated in correlation to the proliferating stage of cells, as observed with DNA polymerase alpha.

T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.

T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.

Inhibition of DNA synthesis by an electrophilic metabolite of benzo[a]pyrene.

Mitogen-stimulated scheduled DNA synthesis and DNA excision repair in human lymphocytes, as well as DNA polymerase a activity in a cell-free system, were inhibited by an electrophilic metabolite of benzo[a]pyrene. This metabolite, (+/-)-anti-(7r,8t)-dihydroxy-(9,10t)-epoxy-7,8,9,10-tetrahyd robenzo[a]pyrene (BPDE), covalently binds to cellular macromolecules and is mutagenic, carcinogenic, and cytotoxic. Human lymphocytes treated with BPDE at concentrations greater than 500-800 ng/ml showed decreases in both mitogen-stimulated DNA synthesis and excision repair of damaged DNA but did not exhibit overt cytotoxicity (excluded trypan blue and maintained an adenylate charge of greater than 0.7). Formation of, and total concentration of, BPDE-DNA adducts was not correlated with inhibition of DNA synthesis. DNA polymerase alpha studies using a cell-free system showed that enzymatic activity was not diminished when purified polymerase was treated with BPDE prior to the addition of template DNA. When the template DNA concentration was varied, BPDE inhibition of enzyme activity was uncompetitive. BPDE inhibition of enzyme activity was found to be noncompetitive when concentrations of dATP, dCTP, or dTTP were varied and competitive when the concentration of dGTP was varied. The data indicate that BPDE competitively inhibits interaction of dGTP with the template-DNA polymerase alpha complex.

Association of diadenosine 5‘,5“‘-P1,P4-tetraphosphate binding protein with HeLa cell DNA polymerase alpha.

An electrophoretically homogeneous high molecular weight form (640,000) of HeLa cell DNA polymerase alpha was shown to catalyze DNA synthesis with a variety of di- and oligoriboadenylates and oligodeoxyriboadenylates as primers with poly(dT) as template. Diadenosine 5',5"'-P1,p4-tetraphosphate (Ap4A) can be utilized as a primer with poly(dT) as template and was found to be covalently attached to the 5'-end of the poly(dA) product. An Ap4A binding protein is tightly associated with the high molecular weight form of DNA polymerase alpha. This protein which exhibits high affinity, noncovalent binding of Ap4A is resolved from the multiprotein DNA polymerase alpha complex, along with other accessory proteins, by hydrophobic affinity chromatography on phenyl-Sepharose columns.