Association of DNA primase with the beta/gamma subunits of DNA polymerase alpha from Drosophila melanogaster embryos

The DNA polymerase and primase activities of the intact DNA polymerase alpha from early embryos of Drosophila melanogaster co-sediment in native glycerol gradients. However, the activities are separated in glycerol gradients containing 2.8 M urea after treatment of the enzyme with 3.4 M urea. The 182,000-dalton alpha subunit which is required for DNA polymerase activity (Kaguni, L.S., Rossignol, J.-M., Conaway, R. C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S.A. 80, 2221-2225) is not required for DNA primase activity. Instead, primase activity resides in the 60,000-dalton (beta) and/or the 50,000-dalton (gamma) subunit. Neither polymerase nor primase has been found in association with the 73,000-dalton polypeptide which co-purifies with the intact enzyme.     

A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae

Biochemical fractionation of the yeast Saccharomyces cerevisiae has revealed a novel DNA primase activity that copurifies with the major DNA polymerase activity. In the presence of RNA precursors and single-stranded DNA (poly(dT), M13), the DNA primase synthesizes discrete length oligoribonucleotides (apparent length, 8-12 nucleotides) as well as longer RNA chains that appear to be multiples of a modal length of 11-12 nucleotides. When DNA precursors are also present, the oligoribonucleotides are utilized by the accompanying DNA polymerase as primers for DNA synthesis. Copurification of these two enzymatic activities suggests their association in a physical complex which may function in the synthesis of Okazaki fragments at chromosomal replication forks.     

Presynapsis and synapsis of DNA promoted by the STP alpha and single-stranded DNA-binding proteins from Saccharomyces cerevisiae

We previously purified an activity from meiotic cell extracts of Saccharomyces cerevisiae that promotes the transfer of a strand from a duplex linear DNA molecule to complementary circular single-stranded DNA, naming it Strand Transfer Protein alpha (STP alpha) (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). This activity requires no nucleotide cofactor but is stimulated more than 10-fold by the addition of yeast single-stranded DNA-binding proteins (ySSBs). In this paper, we describe the aggregation and strand transfer of double-stranded and single-stranded DNA promoted by STP alpha and ySSB. There is a good correlation between the aggregation induced by various DNA-binding proteins (ySSBs, DBPs and histone proteins) and the stimulation of STP alpha-mediated DNA strand transfer. This implies that the stimulation by ySSBs and other binding proteins is probably due to the condensation of single-stranded and double-stranded DNA substrates into coaggregates. Within these coaggregates there is a higher probability of pairing between homologous double-stranded and single-stranded DNA, favoring the initiation of strand transfer. The aggregation reaction is rapid and precedes any reactions related to DNA strand transfer. We propose that condensation into coaggregates is a presynaptic step in DNA strand transfer promoted by STP alpha and that pairing between homologous double- and single-stranded DNA (synapsis) occurs in these coaggregates. Synapsis promoted by STP alpha and ySSBs also occurs between covalently closed double-stranded DNA and single-stranded linear DNA as well as linear double-stranded and linear single-stranded DNAs in the absence of any nucleotide cofactors.     

Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis

Recombinant RF (replicate form) I DNAs containing the bacteriophage phi X174 gene A protein-recognition sequence are cleaved by the phi X A protein yielding a phi X RF II X A protein complex (Zipursky, S.L., Reinberg, D., and Hurwitz, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5182-5186). Such complexes support DNA synthesis in both RF I leads to SS(c) and RF I leads to RF I phi X DNA replication reactions in vitro. Two phi X A protein-recognition sequences were inserted into plasmid pBR322. Both sequences were contiguous with the same strand of the vector DNA and separated by 667 and 4275 base pairs. This recombinant plasmid (G27-4) was cleaved by the phi X A protein at either insert and both inserts support the initiation of RF leads to SS(c) DNA synthesis. This was verified by the finding that replication products were circular molecules of 667 and 4275 nucleotides. This finding is in keeping with the multifunctional activities associated with the phi X A protein; these include the site-specific nicking of RF I DNA which initiates DNA synthesis and site-specific termination resulting in the circularization of the displaced DNA strand. The phi X A protein and the Escherichia coli rep and SSb proteins catalyze the unwinding of phi X RF I DNA in vitro (Scott, J.F., Eisenberg, S., Bertsch, L.L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 193-197). Recombinant plasmid G27-4 RF I DNA was also unwound in vitro by this enzyme system; in this case, both circular and linear single-stranded DNA molecules of 667 and 4275 nucleotides, as well as full length circular single-stranded DNA were formed. Full length linear DNA was not detected. The two single-stranded circular DNA products formed as leading strands in RF leads to SS(c) reaction mixtures containing G27-4 RF I DNA differed in their ability to support lagging strand DNA synthesis. It was shown that the large single-stranded circular product included DNA sequences homologous to a replication factor Y effector sequence required for RF leads to RF and SS(c) leads to RF replication (Zipursky, S.L., and Marians, K.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6521-6525). The 4275-nucleotide, but not the 667-nucleotide, single-stranded circular DNA product was converted to a duplex structure.     

DNA strand transfer protein beta from yeast mitotic cells differs from strand transfer protein alpha from meiotic cells

We have purified to homogeneity an activity from mitotic cell extracts of the yeast Saccharomyces cerevisiae, which promotes the transfer of a strand from a duplex linear DNA molecule to a complementary circular single strand. This activity does not require any nucleotide cofactor and is greatly stimulated by yeast single-stranded DNA-binding protein. It consists of a single polypeptide of an apparent molecular mass of 180 kDa as determined by SDS-polyacrylamide gel electrophoresis. This activity, which we call DNA strand transfer protein beta (STP beta), has reaction properties similar to those of DNA strand transfer protein alpha (STP alpha) purified from crude extracts of yeast meiotic cells (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). However, STP beta differs from STP alpha in its molecular weight and column chromatographic behavior as well as by immunological comparison. Furthermore, the STP beta polypeptide remains in cells in which the STP alpha gene has been disrupted. Thus, we conclude the STP beta activity is encoded by a gene different from that for STP alpha. Although STP beta was isolated from mitotic cells, the amount of STP beta increases severalfold during meiosis. STP beta also appears to differ in molecular weight from similar activities described by other groups and may be an intact form of their activities.     

Purification and properties of a DNA primase from Nicotianatabacum

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase α-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase. Received: 8 May 1997 / Accepted: 5 June 1997     

Immunoaffinity-purified DNA polymerase alpha from a mouse temperature-sensitive mutant, tsFT20 strain, is heat-labile

We have purified DNA polymerase alpha from a temperature-sensitive mutant cell line of mouse FM3A cells, tsFT20, that shows temperature-sensitive activity of DNA polymerase alpha (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1761-1765). The purified enzyme was composed of two polypeptides with the same apparent molecular weights as those of purified DNA polymerase alpha from the parental strain, FM3A (Mr 180,000 and 68,000). Heat inactivation experiments revealed that this purified DNA polymerase alpha from tsFT20 cells was more heat-labile than the wild-type enzyme. We have also purified primase from both ts-FT20 cells and wild-type cells. Both primase fractions consist of two polypeptides with molecular weights of 54,000 and 46,000. No difference was observed between the heat labilities of the primases from tsFT20 cells and wild-type cells. Comparisons of wild-type and mutant polymerase indicated that the temperature-sensitive mutation in DNA polymerase alpha from tsFT20 cells affect the dCTP-binding site of the enzyme. The mutation also changed the optimum pH and the optimum KCl concentration of the enzyme.     

A prepriming DNA replication enzyme of Escherichia coli. II. Actions of protein n': a sequence-specific, DNA-dependent ATPase

Protein n' of Escherichia coli is required for formation of the prepriming complex in replication of the single-stranded circle of phiX174 DNA. The protein, purified to near homogeneity, possesses ATPase (dATPase) activity in the presence of single-stranded, but not duplex, DNAs. Except for phiX174 DNA, ATPase activity is completely suppressed by coating the DNA with single strand binding protein (SSB). phiX174 DNA possesses a unique sequence with a potential hairpin structure that is recognized as an effector (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803). Sequences with secondary structure in SSB-coated M13 DNA which are recognized by RNA polymerase, and in coated G4 DNA by primase, are inert for protein n'. Approximately 30 of the 180 molecules of SSB bound to phiX DNA are destabilized by protein n' in an ATP-dependent reaction. These actions by protein n' may be important in recognizing an origin for forming the prepriming complex that leads to initiation of phiX complementary strand synthesis.     

The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.     

Herpes simplex virus-1 helicase-primase. Physical and catalytic properties.

Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of the products of the UL5, UL8, and UL52 genes (Crute, J. J., Tsurumi, T., Zhu, L., Weller, S. K., Olivo, P. D., Challberg, M. D., Mocarski, E. S. and Lehman, I. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2186-2189). Further characterization of the three-subunit enzyme isolated from HSV-1-infected CV-1 cells shows it to be a heterotrimer, consisting of one polypeptide encoded by each of the UL5, UL8, and UL52 genes. Analysis of the primase and helicase components of the HSV-1 helicase-primase has shown that the primase component synthesizes oligoribonucleotide primers 8-12 nucleotides in length. The helicase component unwinds duplex DNA substrates at the rate of about two nucleotides/s, but only in the presence of the HSV-1-encoded single-stranded DNA binding protein. Thus, the HSV-1 helicase-primase contains the requisite enzymatic activities that permit it to function at the viral replication fork.     

Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.     

Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B.

Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity. To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B. DNA helicase B was purified by sequential chromatography through eight columns. When the purified DNA helicase B was applied to a Mono Q column, the stimulatory activity for DNA primase-catalyzed oligoribonucleotide synthesis and DNA helicase and DNA-dependent ATPase activities of DNA helicase B were co-eluted from the column. The synthesis of oligoribonucleotides 5-10 nt in length was markedly stimulated by DNA helicase B. The synthesis of longer species of oligoribonucleotides, which were synthesized at a low level in the absence of DNA helicase B, was inhibited by DNA helicase B. The stimulatory effect of DNA helicase B was marked at low template concentrations and little or no effect was observed at high concentrations. The mouse single-stranded DNA binding protein, replication protein A (RP-A), inhibited the primase activity of the DNA polymerase alpha-primase complex and DNA helicase B partially reversed the inhibition caused by RP-A.     

A multiprotein form of DNA polymerase alpha from HeLa cells. Resolution of its associated catalytic activities

The majority of the DNA polymerase alpha activity in HeLa cells has been isolated and purified as a multiprotein Mr 640,000 form. The multiprotein form of DNA polymerase alpha corresponds to DNA polymerase alpha 2 that was previously reported by us (Lamothe, P., Baril, B., Chi, A., Lee, L., and Baril, E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4723-4727). The highly purified DNA polymerase alpha 2 has in addition to DNA polymerase alpha-associated DNase, primase, and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A)binding activities and accessory primer recognition proteins C1 and C2. The DNA polymerase alpha and associated activities increase coordinately during the G1/S-phase transition of the cell cycle. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the electrophoretically homogeneous DNA polymerase alpha shows that it is composed of at least eight polypeptides in the molecular weight range of 180,000-15,000. Hydrophobic chromatography on butyl-agarose resolves the DNase and Ap4A-binding protein from a complex of DNA polymerase alpha, primase, and the primer recognition proteins C1 and C2. Hydrophobic chromatography of the latter complex on phenyl-Sepharose resolves the C1 protein from a DNA polymerase alpha-C2 protein-primase complex. Phosphocellulose chromatography of the DNA polymerase-primase-C2 protein complex resolves the C2 protein from a complex of DNA polymerase alpha-primase.     

Signal sequence of alkaline phosphatase of Escherichia coli.

The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.     

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.     

Mechanisms of error discrimination by Escherichia coli DNA polymerase I

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher than that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 10(4) demonstrates the extremely high specificity of base selection by DNA polymerase I and is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W. S., Downey, K. M., & So, A. G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7378]. Studies on the effects of phosphate ion on the polymerase and 3'- to 5'-exonuclease activities of DNA polymerase I showed that, whereas the polymerase activity was somewhat stimulated by phosphate, the exonuclease activity was markedly inhibited, being 50% inhibited at 25 mM phosphate and greater than 90% inhibited at 80 mM phosphate. Selective inhibition of the exonuclease activity by phosphate also resulted in inhibition of template-dependent conversion of a noncomplementary dNTP to dNMP and, consequently, markedly affected the kinetic constants for insertion of noncomplementary nucleotides. The mutagenic metal ion Mn2+ was found to affect error discrimination by both the polymerase and 3'- and 5'-exonuclease activities of DNA polymerase I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.     

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.     

Yeast DNA primase and DNA polymerase activities. An analysis of RNA priming and its coupling to DNA synthesis

The yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates (Singh, H., and Dumas, L. B. (1984) J. Biol. Chem. 259, 7936-7940). In the presence of ATP substrate and poly(dT) template, the enzyme preparation synthesizes discrete-length oligoribonucleotides (apparent length 8-12) and multiples thereof. The unit length primers are the products of de novo processive synthesis and are precursors to the synthesis of the multimers. Multimeric length oligoribonucleotides are not generated by continuous processive extension of the de novo synthesis products, however, nor do they arise by ligation of unit length oligomers. Instead, dissociation and rebinding of a factor, possibly the DNA primase, results in processive extension of the RNA synthesis products by an additional modal length. Thus, catalysis by the yeast DNA primase can be viewed as repeated cycles of processive unit length RNA chain extension. Inclusion of dATP substrate results in three distinct transitions: (i) coupling of RNA priming to DNA synthesis, (ii) suppression of multimer RNA synthesis, and (iii) attenuation of primer length. The less than unit length RNA primers appear to result from premature DNA chain extension, not degradation from either end of the unit length primer. We discuss possible roles of DNA polymerase and DNA primase in RNA primer attenuation.     

Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis.

The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.