Selective inhibition of DNA polymerase alpha by a polysaccharide purified from slime of Physarum polycephalum

A polysaccharide was purified from the slime of a myxomycete, Physarum polycephalum, and its inhibitory effect on eukaryotic DNA polymerases was examined. Almost all the calf thymus DNA polymerase alpha activity was inhibited with higher than 0.2 mg/ml of the polysaccharide, when the assay was carried out with activated DNA as a template. The inhibitory effect occurred regardless of the amounts of the enzyme and deoxyribonucleotides, however, kinetic analysis revealed that the inhibition occurs competitively with the template DNA, the Ki value being 4 micrograms/ml. Inhibition was observed for DNA polymerase alpha, but not for DNA polymerases beta and gamma from various eukaryote species.     

Chick-embryo DNA polymerase gamma. Identity of gamma-polymerases purified from nuclei and mitochondria

The level of DNA polymerase gamma as compared to DNA polymerases alpha and beta has been determined in chick embryo by means of specific tests: the amount of gamma-polymerase in the 12-day-old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase gamma is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment. The gamma-polymerases have been purified from chick embryo nuclei and mitochondria 500-700 times by means of DEAE-cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase gamma is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified gamma enzymes are distinct from the chick embryo DNA polymerases alpha and beta and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial gamma-polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.     

Differential inhibition of various mammalian DNA polymerase activities by ammonium 21-tungsto-9-antimoniate (HPA23)

The 21-tungsto-9-antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase alpha and murine DNA polymerase gamma. HPA23 inhibited the activity of DNA polymerase alpha in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template.primer. The Ki of murine DNA polymerase alpha for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase gamma also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to substrate, dTTP. DNA polymerase beta and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.     

Template specific inhibitor of mammalian DNA polymerases.

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.     

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.     

Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid.

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.     

Comparison among DNA polymerases 1, 2 and 3 from maize embryo axes. A DNA primase activity copurifies with DNA polymerase 2

Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strenghtening the suggestion that polymerase 2 is a replicative enzyme, of the -type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature.     

Study of dogfish (Scyliorhinus caniculus) deoxyribonucleic acid polymerase alpha and beta. Extraction, separation, characterization and changes during spermatogenesis.

DNA polymerase activity was extracted from testis cells of the dogfish Scyliorhinus caniculus. On a sucrose gradient, two main peaks could be separated, corresponding to DNA polymerases beta (3.8 S) and alpha (7.5 S). DNA polymerase gamma could also be detected when poly(A) . (dT)12 was used as template. The properties of alpha and beta polymerases of this primitive vertebrate were similar to those generally described, especially in mammals. The beta enzyme was highly sensitive to N-ethylmaleimide, however, and could use poly(dT) . poly(A) as template. Polymerase alpha was present in spermatogonia, spermatocytes and spermatids. Activity was maximal in spermatocytes. DNA polymerase beta was present in all testis cells with similar activities in spermatogonia and spermatocytes. Decreased activities were observed during spermiogenesis. Some activity remained associated with the chromatin fraction of mature sperm cells.     

A low molecular weight DNA polymerase from wheat embryos

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase C_I) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase C_I did not inhibit animal DNA polymerases and or wheat DNA polymerase A, whereas wheat DNA polymerases C_II and B were much less affected than the C_I enzyme. Several properties of enzyme C_I were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase C_I while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase C_I can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase . The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase , an enzyme believed to be involved in nuclear DNA repair.     

In vitro synthesis of short DNA pieces by DNA polymerase alpha from mouse myeloma

The DNA chain elongation mechanisms of mouse DNA polymerases alpha and beta have been analyzed by using denatured DNA with a (dT)n block at the 3'-end as a template in combination with RNA ((rA)12-20)primer. The (rA)12-20-primed DNA product synthesized by DNA polymerase alpha was 3-5 s in size even after prolonged reaction; instead of a size increase, the number of 3-5 s molecules increased with the reaction time. The size of products was not affected by differences in 3H-labeled substrate (dATP or dTTP), enzyme amount, KCl concentration, or the length of 3'-(dT)n blocks. On the other hand, DNA polymerase beta synthesized long DNA products by a highly distributive reaction mechanism. 3-5 sDNA pieces synthesized by DNA polymerase alpha were not elongated any further by DNA polymerase alpha, but were converted into long DNA chains by DNA polymerase beta. The results imply that DNA polymerase alpha recognizes the size of the product DNA, and shuts off further elongation.     

Three cytoplasmic DNA polymerases that utilize poly(rA).oligo(dT)

Three DNA polymerases that use poly(rA).oligo(dT) were partially purified from cytoplasmic extracts of cultured mouse cells (after removal of mitochondria), and characterized. One is similar to, and may be the same as, the mitochondrial DNA polymerase gamma. The other two enzymes, one 7.5 S and the other 3.6 S, share some properties with DNA polymerases beta and gamma, e.g. their responses to certain inhibitors; however, they are not clearly identified with any previously well-characterized mammalian DNA polymerases. It is also demonstrated that the response of DNA polymerase gamma to N-ethylmaleimide is template dependent, and that DNA polymerase alpha has an authentic (albeit small) activity with poly(rA).oligo(dT).     

Difference in the mechanisms of poly(dT) synthesis by DNA polymerases beta and gamma.

Poly(dT) products which were synthesized depending on (rA)n . (dT)12-18 as a template . primer by mammalian DNA polymerases beta and gamma were analyzed by alkaline sucrose gradient centrifugation. The size of the population of poly(dT) chains synthesized by DNA polymerase beta increased slowly and consistently during incubation up to at least 30 min. On the other hand, the product size with DNA polymerase gamma reached the final size (7 s) within 5 min and the number of products increased during further incubation. Comparison of product number per enzyme molecule suggests that DNA polymerase beta acts on multiple primers in a distributive fashion while DNA polymerase gamma completes poly(dT) chains of large size in a one-by-one fashion.