Inhibition of DNA polymerase activity by thymine hydrates.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.     

Mechanism of ribonucleic acid chain initiation. 1. A non-steady-state study of ribonucleic acid synthesis without enzyme turnover

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.     

Inhibition of ribonucleases by ribonucleotides and transition state analogs in cell-free extracts from Ehrlich ascites tumor cells.

We investigated the ribonucleolytic breakdown of poly(U), poly(A), RNA trascribed from calf thymus DNA with E. coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obrained from a postribosomal supernatant of Ehrlich ascites tumor cells. The single-stranded homopolyribonucleotides are preferentially degraded by the enzyme fraction with the production of ribonucleoside 5'-monophosphates. The RNase activity is completely dependent on the presence of Mg2+ ions and is highest at Mg2+ and K+ concentrations optimal for cell-free protein synthesis. Ribonucleoside 5'-monophosphates, ribonucleoside 2'(3')-monophosphates, ribonucleoside 2'(3'),5'-bisphosphates and transition state analogs consisting of vanadyl sulfate and either ribonucleosides or ribonucleoside 5'-monophosphates in a molar ratio 1:1 inhibit the ribonucleolytic activity of the enzyme fraction. The ribonucleoside 2'(3'),5'-bisphosphates and the transition state analogs are the most effective inhibitors. However, only in the presence of ribonucleoside 2'(3'),5'-bisphosphates a concomitant stimulation by 50 to 60% of poly(U)-directed polyphenylalanine synthesis is observed; all the other RNase inhibitors tested also inhibit polypeptide synthesis. The results of preliminary experiments show that poly(U) and ribonucleoside 2'(3'),5'-bisphosphates are well suited as ligands for affinity chromatography of ribonucleases from Ehrlich ascites tumor cells.     

Alteration of the exonuclease activities of DNA polymerase I by captan

DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.     

Influence of cyclic AMP on DNA synthesis in slices of regenerating rat liver.

DNA synthesis in slices of regenerating rat liver is inhibited by adenosine cyclic 3',5'-monophosphate [cAMP]. The number of cells synthesizing DNA as assayed by 2-14C-thymidine incorporation is reduced by 65% in the presence of 10(-3) M cAMP. The inhibition of cAMP is not specific; other adenosine compounds, N6,O2,-dibutyryl adenosine 3',5'-monophosphate, 5'AMP and adenosine have the same effect. Moreover, the concentration of cAMP in the cell required for this inhibition is much higher than the normal levels of cAMP in liver cells.     

Independent regulation of the nuclear and mitochondrial DNA synthesis induced by either Simian virus 40 or serum in mouse fibroblast 3T3 cells.

Resting cultures of 3T3 cells (an established line of mouse fibroblasts) were released from density inhibition by either infection with Simian virus 40 or addition of serum. The increased rate of thymidine incorporation into DNA, induced by these two agents, was measured in the presence and in the absence of three inhibitory conditions (cycloheximide or dibutyryladenosine 3':5'-monophosphate added to the medium, or lack of anchorage). The inhibition was found to be quite similar in cultures stimulated by virus or serum; under the same conditions, however, the incorporation into mitochondrial DNA was much less inhibited than that into nuclear DNA. The experiments also suggest that new protein synthesis may not be necessary, for either virus or serum, to start the inductive mechanism.     

T7 gene 6 exonuclease has an RNase H activity.

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.     

Fidelity of deoxyribonucleic acid polymerases from normal and leukaemic human cells in polydeoxynucleotide replication (Short Communication)

Although the relative incorporation of incorrect nucleotide (dCTP or dGTP) into poly(dA-dT).poly(dA-dT) by partially purified 3-4S DNA polymerase from normal or leukaemic human cells was four to five times higher than that by the 6-7S DNA polymerase, no significant differences in the infidelity of these polymerases between normal and leukaemic cells were noted.     

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.     

Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.     

Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma

Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.     

Effects of bisulfite (sulfur dioxide) on DNA synthesis and fidelity by DNA polymerase I

In an attempt to explain the mechanism of comutagenesis by bisulfite, the extent and accuracy of DNA synthesis by E. coli DNA polymerase I was examined in the presence of sodium bisulfite. Bisulfite concentration of 100 mM caused nearly complete inhibition of dNTP incorporation into activated calf thymus DNA. Other salts (NaCL, Na2SO4) at the same concentration had no effect on enzyme activity. Preincubation of the various DNA synthesis assay components in 100 mM bisulfite showed that only preincubation of DNA polymerase I caused inhibition of DNA synthesis. Exonuclease functions of DNA polymerase I were unaffected by up to 100 mM bisulfite. Accuracy of DNA synthesis in the presence of bisulfite was determined using poly (dA-dT) as a template-primer. Concentrations of bisulfite greater than 50 mM caused a progressive decrease in enzyme accuracy. At 100 mM bisulfite there was an approximate 7.5-fold decrease in the fidelity of DNA synthesis, compared to control values, as measured by the ratio of noncomplementary (dGTP) to complementary (dTTP) nucleotide incorporated. Based on the known chemistry of bisulfite, it is hypothesized that sulfitolysis of the one disulfide group in DNA polymerase I by bisulfite might be responsible for the reduced polymerase activity and accuracy. The exonuclease functions of DNA polymerase I do not seem to require the disulfide linkage. These results suggest that the effects of bisulfite on mutation frequency might be mediated by effects on the fidelity of DNA repair systems.     

Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.     

The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.

DNA polymerase gamma from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and chloroquine inhibition is less pronounced. In the case of DNA polymerases alpha and beta very little inhibition by ethidium bromide was observed. Intercalation of this dye in a poly dA-dT 12-18 template-primer was studied spectrophotometrically under conditions similar to those in the in vitro DNA polymerase assay. The polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase gamma activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT 12-18. We conclude that the drug, most probably in the intercalated form, is able to interact with the active site (s) of mitochondrial DNA polymerase.     

Purification and characterization of porcine liver DNA polymerase gamma: utilization of dUTP and dTTP during in vitro DNA synthesis.

Porcine liver DNA polymerase gamma has been demonstrated to preferentially incorporate dTMP over dUMP during in vitro DNA synthesis. When polymerase activity was measured in standard reactions containing saturating levels of either dTTP or dUTP, the polymerization rate was slightly faster in the reaction containing dTTP. However, under conditions where both dTTP and dUTP competed, at an equal molar concentration, approximately 3-times more thymine residues were incorporated than uracil residues into DNA. Similarly, preferential incorporation of dTMP was observed on several substrates including poly (dA).oligo p(dT), poly (rA).oligo p(dT) and poly (dA-dT). The discrimination against dUMP incorporation was even more apparent with reduced levels of dUTP. These observations were consistent with the finding that the Km for DNA polymerase gamma was about 3-fold lower for dTTP (0.4 microM) than for dUTP (1.1 microM). On the other hand, the Vmax for these two reactions was very similar. Discrimination against dUMP incorporation was also observed during inhibition of polymerase gamma by dideoxyribonucleoside triphosphates. Dideoxythymidine triphosphate preferentially inhibited dUMP incorporation compared to that of dTMP, whereas ddATP, ddCTP and ddGTP inhibited both reactions equally.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

In vitro conversion of MVM parvovirus single-stranded DNA to the replicative form by DNA polymerase alpha from Ehrlich ascites tumour cells.

A partially purified preparation of DNA polymerase alpha, obtained from the cytosol of Ehrlich ascites tumour cells, has been found to catalyze the conversion of MVM parvovirus, SS DNA (5 kilobases) to RF in vitro. The reaction initiates at a natural 55 base pair hairpin which exists at the 3' terminus of MVM SS DNA. The SS leads to RF conversion is sensitive to aphidicolin, resistant to ddTTP and is promoted by purine ribonucleoside 5' triphosphates, a phenomenon which could not be explained simply by stabilization effects on the in vitro deoxynucleotide precursor pool. In the absence of rNTPs, nascent complementary strands frequently terminate prematurely at a preferred location, between 1300 and 1700 nucleotides from the initiating 3' hairpin terminus. This in vitro system, involving self-primed parvovirus DNA synthesis, provides a convenient assay for those components of the mammalian replicative DNA polymerase complex which are required for the elongation of nascent DNA chains.     

Requirements for synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and gene 4 protein of bacteriophage T7.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze DNA synthesis on duplex DNA templates. Synthesis is initiated at nicks in the DNA template, and this leading strand synthesis results in displacement of one of the parental strands. In the presence of ribonucleoside 5'-triphosphates the gene 4 protein catalyzes the synthesis of oligoribonucleotide primers on the displaced single strand, and their extension by T7 dna polymerase accounts for lagging strand synthesis. Since all the oligoribonucleotide primers bear adenosine 5'-triphosphate residues at their 5' termini, [gamma 32P]ATP is incorporated specifically into the product molecule, thus providing a rapid and sensitive assay for the synthesis of the RNA primers. Both primer synthesis and DNA synthesis are stimulated 3- to 5-fold by the presence of either Escherichia coli or T7 helix-destabilizing protein (DNA binding protein). ATP and CTP together fully satisfy the requirement for rNTPs and provide maximum synthesis of primers and DNA. Provided that T7 DNA polymerase is present, RNA-primed DNA synthesis occurs on either duplex or single-stranded DNA templates and to equal extents on either strand of T7 DNA. No primer-directed DNA synthesis occurs on poly(dT) or poly(dG) templates, indicating that synthesis of primers is template-directed.     

A study on the unprimed poly (dA-dT) synthesis catalyzed by preparations of E. coli DNA polymerase I.

Evidence was obtained indicating that the initiation of poly (dA-dT) de novo synthesis is provided by deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase present in preparations of E. coli DNA polymerase I and capable of catalyzing the unprimed polymerization of dNDP. dNDP-transferase synthesyzes short oligonucleotides which form template-primer complexes repeatedly replicated by DNA polymerase I. This conclusion was based on the following observations: the abolition of the lag period of poly (dA-dT) synthesis by preincubation of DNA-polymerase I preparations with dADP and dTDP; the presence of oligo (dA-dT) among the preincubation products; the suppressive effect of dithiothreitol and N-ethylmaleimide (inhibitors of dNDP-transferase) on the de novo, but not on the primed synthesis of poly (dA-dT), catalyzed by preparations of DNA-polymerase I.