Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.     

DNA polymerase I and DNA primase complex in yeast

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.     

Initiation of enzymatic DNA synthesis by yeast RNA polymerase I.

In vitro DNA synthesis by yeast DNA polymerase I can be initiated by partially purified yeast RNA polymerases in the presence or absence of rNTPs. Homogeneous yeast RNA polymerase I initiates DNA synthesis by yeast DNA polymerase I on single-stranded DNA templates only in the presence of all four rNTPs. A protein capable of initiating enzymatic DNA synthesis on single-stranded DNA in the absence of rNTPs has also been separated from partially purified yeast RNA polymerase I fractions. Analysis of the RNA polymerase I initiated replication products of phage fd DNA on alkaline sucrose gradients showed noncovalent linkage between the newly synthesized DNA and the template. Isopycnic analyses of the ribonucleotide initiated fd DNA replication products demonstrated covalent linkage between the initiator RNA and newly synthesized DNA. Results from 32P-transfer experiments confirmed the covalent linkage between RNA and DNA chains and showed the presence of all four ribo- and deoxyribonucleotides at the RNA--DNA junctions. The ribonucleotide found most frequently at the RNA--DNA junction is uridylate and the purine deoxynucleotides occur more frequently than pyrimidine deoxynucleotides.     

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.     

DNA primase isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.     

Characterization of DNA primase separated from DNA polymerase alpha-DNA primase complex of calf thymus

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.     

Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.     

RNA and DNA synthesis catalyzed by a DNA-polymerase alpha complex with primase from silkworm cells on single-stranded DNA]

Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation.     

Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase.

DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10(-6) and 10 x 10(-6). This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex

An immunoaffinity chromatographic procedure was developed to purify DNA polymerase-DNA primase complex from crude soluble extracts of yeast cells. The immunoabsorbent column is made of mouse monoclonal antibody to yeast DNA polymerase I covalently linked to Protein A-Sepharose. Purification of the complex involves binding of the complex to the immunoabsorbent column and elution with concentrated MgCl2 solutions. After rebinding to the monoclonal antibody column free primase activity is selectively eluted with a lower concentration of MgCl2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of five major peptides, p180, p140, p74, p58, and p48 in the immunoaffinity-purified DNA polymerase-DNA primase complex. Free primase and free polymerase fractions obtained by fractionation on the immunoabsorbent column were analyzed on activity gels and immunoblots. These analyses showed that p180 and p140 are DNA polymerase peptides. Two polypeptides of 58 and 48 kDa co-fractionated with the free yeast DNA primase. From sucrose gradient analysis we estimate a molecular weight of 110 kDa for the native DNA primase.     

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase alpha-primase.

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase alpha-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase alpha-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

Thymine glycol lesions terminate chain elongation by DNA polymerase I in vitro.

Single-strand circular DNA from bacteriophage M13mp9 was chemically modified with osmium tetroxide to introduce specifically cis-thymine glycol lesions, a major type of DNA damage produced by ionizing radiation. An oligonucleotide primer was extended on damaged and undamaged templates using either the large fragment of E. coli pol I or T4 DNA polymerase. The reaction products were analysed by electrophoresis alongside a DNA sequence ladder. Synthesis on the damaged templates terminated at positions opposite thymine bases in the template. These results indicate that cis-thymine glycol lesions in single-strand DNA constitute blocks to synthesis by DNA polymerases in vitro. Surprisingly, replication halts after the correct nucleotide, dAMP, is inserted opposite the lesion. These results imply that the primary effect of the thymine glycol lesion is suppression of DNA synthesis and that the lesion is not a potent mutagen.     

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.     

DNA primase. Processivity and the primase to polymerase alpha activity switch

Calf thymus DNA primase was examined to determine the kinetic parameters that define its unusual processivity. At 37 degrees C, the major products were 8-9 and 2-3 nucleotides long. The 2-mer was the predominant product when considered on a molar basis. At each polymerization cycle en route to synthesis of a unit length primer (7-10 nucleotides), processivity was defined by competition of enzyme dissociation with ATP binding as well as an ATP independent step(s). Reducing the temperature to 25 degrees C had relatively little effect on the production of primers less than or equal to 6 nucleotides long, but greatly enhanced production of primers twice (16-18 nucleotides) the normal unit length. Kinetic analysis revealed that synthesis of these longer primers largely involves dissociation of the primase after completion of the unit length primer. After synthesis of a primer, the primase-polymerase complex normally switches to polymerase activity. Only primers greater than or equal to 7 nucleotides long were utilized by the polymerase regardless of the dNTP concentration, indicating that the signal for the primase to polymerase activity switch is primer completion. During the switch, either the primer-template does not dissociate from the complex or the complex has extraordinarily high affinity for the primers. At 25 degrees C and physiological dNTP concentrations the activity switch is very efficient, greater than 90% of the primers are elongated. However, at 37 degrees C the switch is much less efficient, likely due to primer-template denaturation.     

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.     

Initiation of chromosomal DNA synthesis in vitro

Summary An in vitro complementation assay for initiation of chromosomal DNA replication is described. The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains. Each hybrid plasmid carries a suppressor of dnaA-ts mutations. The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.