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.     

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.     

Modified polynucleotides. VII. Impaired integrity of a synthetic DNA containing the antiherpetic agent 5-isopropyl-2''-deoxyuridine.

5-Isopropyl-2'-deoxyuridine (ip5dU) was recently recognized as a clinically useful antiherpetic (HSV-1) agent. An ip5dU-containing polynucleotide, poly (dA-dT, ip5dU) was prepared to study how physical and bio-organic properties of the synthetic DNA model poly (dA-dT) would change upon partial substitution of thymidine. Synthesis was carried out with DNA polymerase enzyme and the polymers contained 7-9% of ip5dU. It proved to be less thermostable than poly (dA-dT) and the transition width was highly increased. Although it was a very efficient template for DNA polymerase enzyme, template activity for RNA synthesis was strongly reduced by the presence of ip5dU. Diminished stability against enzymic degradation, especially against single-strand-specific Nuclease S1 was also observed.     

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.     

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.     

Pathways of assimilation of [13N]N2 and 13NH4+ by cyanobacteria with and without heterocysts.

Daughter strand gaps are secondary lesions caused by interrupted DNA synthesis in the proximity of UV-induced pyrimidine dimers. The relative roles of DNA recombination and de novo DNA synthesis in filling such gaps have not been clarified, although both are required for complete closure. In this study, the Escherichia coli E486 and E511 dnaE(Ts) mutants, in which DNA polymerase I but not DNA polymerase III is active at 43 degrees C, were examined. Both mutants demonstrated reduced gap closure in comparison with the progenitor strain at the nonpermissive temperature. These results and those of previous studies support the hypothesis that both DNA polymerase I and DNA polymerase III contribute to gap closure, suggesting a cooperative effort in the repair of each gap. Benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography analysis for persistence of single-strand DNA in the absence of DNA polymerase III activity suggested that de novo DNA synthesis initiates the filling of daughter strand gaps.     

Mechanism of inhibition of deoxyribonucleic acid synthesis by 1-beta-D-arabinofuranosyladenosine triphosphate and its potentiation by 6-mercaptopurine ribonucleoside 5'-monophosphate

The mechanism of inhibition of DNA synthesis by 1-beta-D-arabinofuranosyl-ATP (ara-ATP) and the potentiation of this inhibition by 6-mercaptopurine ribonucleoside 5'-monophosphate (6-MPR-P) have been investigated with mammalian DNA polymerase delty by using poly(dA-dT) as the template. The inhibition of DNA synthesis by ara-ATP correlates with incorporation of ara-AMP into poly(dA-dT). Nearest-neighbor analysis indicates that ara-AMP does not act as an absolute chain terminator but rather that chains with 3'-terminal arabinosyl nucleotides are extended slowly. The inhibition of DNA synthesis by ara-ATP is markedly enhanced by the addition of the nucleotide derivative of 6-mercaptopurine, 6-mercaptopurine ribonucleoside 5'-monophosphate. The increased inhibition of DNA synthesis in the presence of 6-MPR-P is due to increased incorporation of ara-AMP. The mechanism by which 6-MPR-P increases the incorporation of ara-AMP is by selective inhibition of the 3' to 5' exonuclease activity of DNA polymerase, thereby preventing the removal of newly incorporated ara-AMP at 3' termini of DNA chains.     

De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase.

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed in Escherichia coli. The truncated fusion protein (NS5Bdelta24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus- or minus-strand viral RNA synthesis de novo in a primer-independent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA-dependent RNA polymerase in vitro. Both Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in the presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mm GTP, and a 3'-terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore, the purified protein was also shown, by North-Western blot analysis, to specifically interact with the 3'-end of both plus- and minus-strand viral RNA templates.     

Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2''-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.     

The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases

N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.     

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.     

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.     

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.     

Metabolic consequences of DNA damage: alteration in purine metabolism following poly(ADP ribosyl)ation in human T-lymphoblasts

The effect of DNA damage caused by N-methyl-N'-nitro-nitrosoguanidine (MNNG) on poly(ADP-ribose) synthesis, NAD levels, and purine nucleotide metabolism was studied in human T-lymphoblasts. Excessive DNA breaks caused by MNNG activated poly(ADP-ribose) polymerase and rapidly consumed intracellular NAD. NAD depletion was followed by rapid catabolism of ATP as well as induction of total purine nucleotide catabolism leading to excretion of purine catabolic products. MNNG-treated cells were not able to replenish the intracellular nucleotide pools due to the depletion of intracellular ATP and phosphoribosylpyrophosphate pools which are required for de novo purine biosynthesis. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide prevented both the depletion of NAD pools and the associated changes in purine nucleotide metabolism.     

The chromatographic properties of the DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]

The DNA-dependent DNA-polymerase (DNA polymerase I which is not sorbed on the column with DEAE-cellulose, and DNA-polymerase II, which is absorbed by this column and is eluted from it by 0.3 M of NaCl), have been isolated from Acholeplasma laidlawii PG-8. DNA-polymerase I in homogeneous state was obtained as a result of the stepwise treatment by heparin-sepharose (elution at 0.35 M of NaCl) and poly-U-sepharose (elution at 0.3 M of NaCl). It was presented on the electrophoregram by one polypeptide with molecular weight of 72 kDalton. The second form of DNA polymerase was also obtained in homogeneous state as a result of sequential treatment on heparin-sepharose (elution at 0.3 M of NaCl) and on poly-A-sepharose (elution at 0.25 M of NaCl): the protein which had manifested polymerase activity was a polypeptide with molecular weight of 45 kDalton.