Ribonucleotidyl transferase in preparations of partially purified DNA polymerase alpha of the sea urchin.

Three ribonucleotidyl transferase types have been described in the sea urchin: riboadenylate trnasferase, the DNA dependent RNA polymerases, and a DNA polymerase associated ribonucleotidyl transferase (Biochemistry 15:3106-3113, 1976). In the present work this latter ribonucleotidyl transferase was found to purify with DNA polymerase alpha through phosphocellulose, DEAE-Sephadex and DNA cellulose and to cosediment at 6.5 S. This ribonucleotidyl transferase was active with Mn+2, but not Mg+2, on calf thymus DNA and poly(dC). Other synthetic templates elicited DNA polymerase alpha but no ribonucleotidyl transferase activity. From alkaline hydrolysates of the poly(dC) directed GTP polymerization, we found Goh and Gp in a ratio of 1:16 indicating an average chain length of 17 residues after a 20 min reaction. Co-polymerization of GTP (5 micrometer) and dGTP (10 micrometer) yielded a non-random distribution of the ribonucleotide in the deoxyribonucleotide. The properties of this urchin ribonucleotidyl transferase are unlike any previously described eukaryotic transferase and the data is discussed with reference to the known properties of E. coli DNA polymerase I and the primase.     

Localization of DNA polymerase alpha on the nuclear membrane in sea urchin embryos

Subnuclear localization of DNA polymerase alpha was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase alpha. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm3) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase alpha associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase alpha was able to associate with nuclear DNA polymerase alpha from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase alpha associates in unfertilized eggs.     

DNA polymerase beta

Mammalian DNA polymerase beta(beta-pol) is a single polypeptide chain enzyme of 39kDa. beta-pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of beta-pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the beta-pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of beta-pol's role in protecting genomic DNA.     

Synthesis of sulfoquinovosylacylglycerols, inhibitors of eukaryotic DNA polymerase alpha and beta

Sulfoquinovosyldiacylglycerols (SQDGs) and sulfoquinovosylmonoacylglycerols (SQMGs), bearing diverse fatty acids, were synthesized from D-glucose, and were examined for enzymatic inhibitions of DNA polymerase alpha and beta. These results indicated that the carbon numbers of the fatty acids were highly related to the activities, at least in vitro, of eukaryotic DNA polymerase inhibition.     

Isolation and various properties of beta DNA polymerase from the embryos of the sea urchin Strongylocentrotus intermedius

DNA-polymerase beta was isolated from embryonic cells of the sea urchin S. intermedius and purified 1040-fold. The molecular weight of the enzyme is 40 000, sedimentation coefficient 3.2S, pI 8.5. The SH-reagent--N-ethylmaleimide--has no appreciable influence on the enzyme activity. The enzyme is thermolabile and needs four deoxyribonucleoside triphosphates, bivalent metal ions (Mg2+ or Mn2+) and primer template for its activity. The maximal activity is observed when a synthetic polymer--poly(dA).oligo(dT) is used. DNA-polymerase performs DNA synthesis via a distributive mechanism. In terms of physico-chemical properties, the enzyme can be related to DNA-polymerases beta.     

Restriction fragment primed phi X174 single-stranded DNA as template for DNA polymerase alpha and beta. Detection and partial purification of a polymerase alpha stimulating factor

Template-primers constructed of phiX174 single-stranded viral DNA hybridized to a restriction fragment of phiX174 RF DNA can be used for extensive polymerization by DNA polymerase alpha. Polymerization is dependent upon a restriction fragment containing a 3'OH. The products of the reaction have been identified by agarose gel electrophoresis. Polymerization of 150--400 nucleotides can be obtained in 1h depending upon the restriction fragment used as primer. Synthesis may be limited by barriers in the primary or secondary structure of the template. A factor which stimulates the rate of alpha polymerase activity on these templates was partially purified. This factor does not stimulate alpha polymerase on activated DNA. The stimulating factor sediments at 5.5 S in glycerol gradients containing 0.4M potassium phosphate and has an apparent molecular weight of 70 000 on Sephadex G-100.     

DNA polymerase alpha, beta and gamma activities in ultraviolet irradiated CV-1 monkey cells.

To determine the possible role of DNA polymerase alpha, beta and gamma during the repair period following ultraviolet (lambda max : 254 nm) irradiation of monkey CV-1 cells, we measured the three enzymatic activities by using specific tests, either in crude extracts or after fractionation by sucrose gradient (5--20%) centrifugation at high salt concentration. When compared to the unirradiated control, we could not detect any significant variation in the levels of activity of DNA polymerases alpha, beta and gamma at any time (0, 12 to 48 h) after ultraviolet irradiation of the cells with doses ranging from 9 to 52.5 J.m-2.     

Mechanism of release of active alpha subunit from dimeric alpha beta avian myeloblastosis virus DNA polymerase.

Storage of the dimeric (alphabeta) form of avian myeloblastosis virus (AMV) DNA polymerase in glycerol resulted in the release of the smaller alpha subunit, as detected by glycerol gradient sedimentation. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme stored in glycerol showed the concomitant appearance of several polypeptides and a lowering in the level of both beta and alpha components. This reduction appears to be the result of cleavages introduced by traces of hydrolytic activity present in glycerol samples. An enhancement of alpha subunit released, as detected by activity profile, was also achieved upon direct but limited exposure of purified avian myeloblastosis virus DNA polymerase to carboxymethyl-cellulose-bound trypsin matrix. Electrophoretic analysis of digested enzyme revealed a progressive fragmentation, with simultaneous increase in the alpha subunit and decrease in the beta subunit.     

DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase.

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.     

Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide.

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.     

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.     

DNA polymerase alpha and models for proofreading.

Using a modified system to measure fidelity at an amber site in phi X174, we have employed DNA polymerase alpha to test different mechanisms for proofreading. DNA polymerase alpha does not exhibit the characteristics of "kinetic proofreading" seen with procaryotic polymerases. Polymerase alpha shows no evidence for a "next nucleotide" effect, and added deoxynucleoside monophosphates do not alter fidelity. Pyrophosphate, which increases error rates with a procaryotic polymerase, appears to weakly improve polymerase alpha fidelity. DNA polymerase alpha does exhibit a dramatic increase in error rate in the presence of a deoxycytidine thiotriphosphate (dCTP alpha S), but this enhanced mutagenesis also occurs under conditions where kinetic proofreading should be otherwise defeated. This particular effect with dCTP alpha S appears specific for DNA polymerase alpha and is not seen with the other polymerases tested.     

Ehrlich ascites tumor (EAT) chalone effects on nascent DNA synthesis and DNA polymerase alpha and beta.

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of 3H-thymidine (3H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50-200 mug/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude alpha- and beta-polymerase activities were inhibited. Crude DNA polymerase for C3H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or 'gapped' DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting alpha- and beta-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating the DNA ligase is not inhibited.     

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 of gamma-like DNA polymerase from sea urchin embryos.

A gamma-like DNA polymerase devoid of DNA polymerase-alpha and -beta activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) . oligo(dT)12--18 followed by poly(dA) . oligo(dT)12--18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-gamma.