Inhibition of DNA polymerase activity by methyl methanesulfonate

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.     

The mode of inhibitory action by aphidicolin on eukaryotic DNA polymerase alpha.

The mode of action by aphidicolin on DNA polymerase alpha from the nuclear fraction of sea-urchin blastulae was studied. The inhibition of DNA polymerase alpha by aphidicolin was uncompetive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template-primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase alpha by competing with dCTP with a Ki value of 0.5 microgram/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC). (dG)12-18 or poly(dA) . (dT)12-18. Similar results were obtained for DNA polymerase alpha (B form) from mouse myeloma MOPC 104E.     

Pα-Methyl Deoxynucleoside Triphosphates as Substrates for E. COLI DNA Polymerase I in a Template-Directed Synthesis of DNA

Abstract

Pα-methyl deoxynucleoside triphosphates are used as substrates for E. coli DNA polymerase I in template-directed polymerase reactions. It is shown that the modified compounds are incorporated together with the unmodified deoxynucleoside triphosphates into DNA under both nick-translation and Klenow reaction conditions.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

Solution structure of a viral DNA repair polymerase.

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP-bound conformation and high binding affinity for dGTP-Mg(2+) of Pol X may contribute to its low fidelity.     

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.     

N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase

Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.     

In vitro repair of UV-or X-irradiated bacteriophage T4 DNA by extract from blue-green alga Anacystis nidulans

The cell-free extract from blue-green alga Anacystis nidulans contains enzymatic activities which repair in vitro transforming DNA of bacteriophage T4 damaged by UV light or X-rays. The repair effect of the extract was observed with double-stranded irradiated DNA but not with denatured irradiated DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture and on the dose of irradiation. A fraction of DNA lesions induced by X-rays is repaired by a NAD-dependent polynucleotide ligase present in the extract. The repair of UV-induced lesions is the most efficient in the presence of magnesium ions, NAD, ATP and the four deoxynucleoside triphosphates. The results indicate that the repair of UV-irradiated DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of the algae on the background of a low deoxyribonuclease activity.Abbreviations UV ultraviolet - TA transforming activity - PN-ligase polynucleotide ligase - NAD nicotinamide adenine dinucleotide - dNTP deoxynucleoside triphosphates - dATP, dGTP, dTTP triphosphates of deoxyadenosine, deoxyguanosine, deoxythymidine and deoxycytidine, respectively     

Inhibition of Escherichia coli DNA polymerase I by rose bengal

The fluorescein dye, rose bengal, inhibits Escherichia coli DNA polymerase I reversibly in the dark and irreversibly in the light. The reversible inhibition, which occurs in the micromolar concentration range, is competitive with respect to the poly(dA-T) template/ primer and noncompetitive with respect to the complementary deoxynucleoside triphosphates. The Hill coefficient for the inhibition by rose bengal is 3.0. Equilibrium dialysis experiments using ¹³¹I-labeled rose bengal have demonstrated direct binding of the inhibitor to the enzyme. No dye binds to poly(dA-T) at concentrations where the inhibition is observed. There are 22 ± 3 rose bengal binding sites per polymerase which can be subdivided into a class of high affinity sites and one of low affinity sites. The high affinity sites (3 μm) bind rose bengal with a Hill coefficient of 1.7 and are responsible for the observed inhibition. The low affinity sites (7μm) are more numerous (about 16) and bind rose bengal in a noncooperative manner. The displacement of rose bengal from the enzyme by poly(dA-T) at equilibrium confirms the competition between poly(dA-T) and rose bengal inferred from the kinetic data for the polymerization reaction. The inhibition of the 3′,5′ exonuclease activity and the template-directed dATP ? P-P exchange reaction by rose bengal is fully consistent with the interaction of rose bengal at the polynucleotide binding site. The enzyme induces an extrinsic Cotton effect in the visible absorption of rose bengal. The abolition of this Cotton effect by poly(dA-T) further supports the proposed site of binding of the dye.     

Effect of seven Indian plant extracts on Fenton reaction-mediated damage to DNA constituents

The influences of substoichiometric amounts of seven plant extracts in the Fenton reaction-mediated damage to deoxynucleosides, deoxynucleoside monophosphates, deoxynucleoside triphosphates, and supercoiled plasmid DNA were studied to rationalize anticancer properties reported in some of these extracts. Extracts from Acacia catechu, Emblica officinalis, Spondias dulcis, Terminalia belerica, Terminalia chebula, as well as gallic acid, epicatechin, chebulagic acid and chebulinic acid enhance the extent of damage in Fenton reactions with all monomeric substrates but protect supercoiled plasmid DNA, compared to standard Fenton reactions. The damage to pyrimidine nucleosides/nucleotides is enhanced by these extracts and compounds to a greater extent than for purine ones in a concentration dependent manner. Dolichos biflorus and Hemidesmus indicus extracts generally do not show this enhancement for the monomeric substrates though they protect plasmid DNA. Compared to standard Fenton reactions for deoxynucleosides with ethanol, the presence of these five plant extracts render ethanol scavenging less effective as the radical is generated in the vicinity of the target. Since substoichiometric amounts of these extracts and the four compounds produce this effect, a catalytic mechanism involving the presence of a ternary complex of the nucleoside/nucleotide substrate, a plant compound and the hydroxyl radical is proposed. Such a mechanism cannot operate for plasmid DNA as the planar rings in the extract compounds cannot stack with the duplex DNA bases. These plant extracts, by enhancing Fenton reaction-mediated damage to deoxynucleoside triphosphates, slow down DNA replication in rapidly dividing cancer cells, thus contributing to their anticancer properties.     

DNA polymerase in nucleoli isolated from Ehrlich ascites tumor cells

DNA replication was investigated in nucleoli isolated from Ehrlich ascites tumor cells. DNA synthesis was dependent on the presence of the four deoxynucleoside triphosphates and magnesium, but was reduced in the presence of ATP. The pH optimum for DNA replication was 8.5 to 9.0 N-Ethyl-maleimide reduced the reaction significantly. DNA synthesis occurred on nucleolar chromatin and was stimulated by treatment of the nucleoli with a small amount of DNase I. Addition of exogenous DNA to the reaction mixture significantly stimulated [³H]dTMP incorporation.     

Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity.

Herpes simplex virus (HSV) DNA polymerase was isolated on a large-scale from African green monkey kidney cells infected with HSV type 1 (HSV-1) strain Angelotti. After DNA-cellulose chromatography the enzyme showed a specific activity of 48,000 units/mg protein. Three major single polypeptides with molecular weights of 144,000, 74,000 and 29,000 were copurified with the enzyme activity at the DNA-cellulose ste. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular alpha, beta and gamma DNA polymerase activities. Two exonucleolytic activities were found in the DNA-cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single-stranded and double-stranded DNA to deoxynucleoside 5'-monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3'-to-5'-exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase delta, the HSV-polymerase-associated exonuclease catalyzed the removal of 3'-terminal nucleotides from the primer/template as well as the template-dependent conversion of deoxynucleoside triphosphates to monophosphates.     

Partial purification of a chloroplast DNA polymerase from Euglena gracilis

A single DNA polymerase has been purified 965 fold from isolated chloroplasts of $\text{Euglena}$$\text{gracilis}$ with a yield of 53%. The isolation methods include solubilization of the enzyme with 1M NaCl, ammonium sulfate precipitation, DNA affinity and DEAE-cellulose chromatography. The enzyme requires all four deoxynucleotide triphosphates, magnesium and denatured DNA for maximal activity. The chloroplast DNA polymerase is free of contaminating nucleases and phosphatases, has a sharp pH optimum at pH 7.2 and magnesium optimum of 6mM.     

Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.

UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide. This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates. Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex. Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I. The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links. The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.     

DNA binding domain of Escherichia coli DNA polymerase I: identification of arginine-841 as an essential residue

To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), we have used an active-site-directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA pol I with PG resulted in the loss of polymerase, 3'-5'-exonuclease, and DNA binding functions. Furthermore, the presence of DNA but not deoxynucleoside triphosphates protected the enzyme from inactivation. Labeling studies with [7-14C]PG indicated that two arginine residues were modified per mole of enzyme. In order to locate the site of PG modification, we digested the PG-treated enzyme with trypsin and V-8 protease. The resulting peptides from each digest were then resolved on reverse-phase hydrophobic columns. An appearance of a new peptide peak was observed in both tryptic and V-8 protease digests. Since inclusion of template-primer during PG modification of enzyme blocks the appearance of these peaks, these peptides were concluded to represent the template-primer binding domain of pol I. Indeed, the extent of inactivation of enzyme by PG treatment correlated very well with the quantitative increase in the new tryptic peptide peak. Amino acid composition analysis of both tryptic peptide and V-8 peptide revealed that the two peptides were derived from the same general region; tryptic peptide spanned between residues 837 and 857 while V-8 peptide spanned between residues 841 and 870 in the primary sequence of pol I. Sequence analysis of tryptic peptide further identified arginine-841 as the site of PG modification, which implicates this residue in the DNA binding function of pol I.     

Incorporation of nucleotides into DNA by mammalian DNA polymerase in the presence of a single deoxynucleoside triphosphate.

Rat ascites hepatoma cell DNA polymerases (EC 2.7.7.7), especially low molecular weight polymerase, could incorporate a significant amount of single nucleotide into acid-soluble products in the absence of the other three deoxynucleoside triphosphates when activated DNA was used as a template. This relaxed requirement for deoxynucleotides was not observed when poly[d(A-T).d(T-A)] was used as a template. Nearest-neighbour base analyses of the products formed in the presence of a single deoxynuclesode triphosphate revealed that the reaction is not of a terminal transferase-type but a very limited repair synthesis in which one or a few triphosphates are incorporated at numerous 3'-hydroxyl ends.     

Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy

The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.     

Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.     

Kinetics of base misinsertion by DNA polymerase I of Escherichia coli

A simple kinetic analysis of the values of kcat and KM for base insertion and misinsertion during DNA replication is presented and applied to the problem of base misinsertion by DNA polymerase I of Escherichia coli. The role of minor tautomeric forms of deoxynucleoside triphosphates (dNTPs) in purine x pyrimidine mismatching has been examined and it has been shown that the misinsertion frequency via this route should be close to the tautomerization constant in solution and is independent of any effect of the polymerase on the tautomerization of a dNTP when bound. Kinetic data on purine x pyrimidine mismatching indicate that the dNTP in a polymerase-DNA-mismatched-dNTP complex is predominantly in the major tautomeric form. The mutagenic effect of Mn2+ in DNA replication is shown to be mediated by decreasing the values of kcat/KM for the insertion of correct dNTPs, whilst the values of this rate constant for misinsertion are relatively unaffected or increased.