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.     

Inhibitory effects of 5-alkyl- and 5-alkenyl-1-beta-D-arabinofuranosyluracil 5'-triphosphates on herpes virus-induced DNA polymerases

Various 5-substituted 1-beta-D-arabinofuranosyluracil 5'-triphosphates (H, methyl, ethyl, n-propyl, n-butyl, (E)-bromovinyl, styryl, and beta-phenylethyl derivatives) were prepared and their inhibitory effects on two different herpes virus-induced DNA polymerases (OMV and HCMV) were studied. These dTTP analogues inhibited the incorporation of [3H]dTMP into DNA in vitro. Among them, analogues having a vinyl group at the 5-position were strongly active against DNA polymerases induced on herpes virus infection. Kinetic analysis showed that the inhibition by the analogues was essentially competitive with respect to the substrate, dTTP. The K1 values (microM) for AraUTP (2.4), AraTTP (1.0), BVAUTP (0.8), and StUAUTP (0.8) were smaller than the Km value (microM) for dTTP (3.4), but those for AraEtUTP, AraPrUTP, and AraBuUTP (5-14) were larger than the Km for dTTP in the case of HCMV-induced DNA polymerase. In contrast to these results, OMV-induced DNA polymerase seemed to be more resistant to these inhibitors than HCMV-induced DNA polymerase. However, the mode of the structure of substituent groups at the 5-position of base moieties is almost the same for the two DNA polymerases, except for in the case of AraUTP itself.     

Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

Mechanism of Inhibition of DNA Synthesis by 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)uracil

The mechanism of the inhibitory action of 1-β-D -arabinofuranosyl-E-5-(2-bromovinyl) uracil triphosphate (BV-araUTP) on DNA synthesis by Escherichia coli DNA polymerase I Klenow fragment was studied. Acting as a chain terminator, BV-araUTP inhibited DNA synthesis by Klenow fragment more effectively than 2′, 3′-dideoxythymidine triphosphate (ddTTP). However, the incorporation sites of BV-araU monophosphate were restricted at consecutive dTMP sequence whereas ddTMP was incorporated at every dTMP site.     

Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases

The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.     

5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2'-deoxy- uridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction.

The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5, 6-tetrafluorobenzoyl)-3-aminopropionyl]aminomethyl}-, and 5-{N-[N'-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2'-de oxyuridine-5'-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication.     

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.     

DNA synthesis in toluene-treated bacteriophage-infected minicells or Bacillus subtilis.

Bateriophage (phi29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 . 10(5)). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the E13H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil.     

Substrate specificity of DNA polymerases. I. Enzyme-catalysed incorporation of 5-''1-alkenyl)-2''-deoxyuridines into DNA.

A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions. Experiments were carried out in E. coli DNA polymerase I Klenow fragment enzyme system. Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer. Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values. Reactivity (Vmax/KM) showed no correlation with the extent of incorporation. 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values. Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept.     

A study of the influence of newly synthesized acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline derivatives on deoxythymidine and deoxycytidine kinase activities in human neurofibrosarcoma and ovarian cancer

The influence of nine newly synthesized uracil acyclonucleosides, and 36 derivatives of 1,2,3,4-tetrahydroisoquinoline on the activity of enzymes catalysing dTMP and dGMP synthesis, on the content of dTTP and dGTP in acid soluble fraction and on the incorporation of [14C]dThd and [14C ]dGuo into DNA in tumour homogenates was studied. The influence of the compounds was studied in the cytosol from intraoperatively excised human tumours - neurofibrosarcoma and ovarian cancer. It was shown that dTMP and dGMP synthesis is inhibited competitively by 34.1+/-4.0% in both types of tumours by 0.2 mM 1-N-(3'-hydroxypropyl)-6-methyluracil (1) and 0.2 mM 1-N-(3'-hydroxypropyl)- 5,6- tetramethyleneuracil (2). The mentioned acyclonucleosides reduced the content of dTTP and dGTP in the acid soluble fraction of tumours (59.7+/-3.1% of control). 1-(4-chlorophenyl)-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (3), 1-(2,3-dichlorophenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (4) and 1-(3-methoxyphenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (5) at 0.2 mM concentration caused a mixed type inhibition of the synthesis of dTMP and dGMP by, on average, 33.2+/-4.4%, and reduced the content of dTTP and dGTP in the acid soluble fraction (52.6+/-3.7% of control) but were active only in the cytosol of neurofibrosarcoma. While acyclonucleosides undergo phosphorylation in the cytosol by cellular kinases, with their triphosphates being active acyclonucleoside metabolites, active 1,3,4,5-tetrahydroisoquinoline derivatives (compounds not containing a deoxyribose moiety), cannot be phosphorylated. ACN and THI derivatives which inhibit dThd and dCyd kinase activities, inhibit also the incorporation of [14C]dThd and [14C]dGuo (ACN - 50.2+/-2.7%, THI - 53.4+/-3.9% of incorporation inhibition) into tumour DNA. The obtained results point to the mechanism of uracil acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline biological activity consisting in inhibiting the synthesis of DNA components.     

Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions.

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K a = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (p K a = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.     

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.     

Deoxythymidine nucleotide metabolism in Bacillus subtilis W23 infected with bacteriophage SP1Oc: preliminary evidence that dTMP in SP10c DNA is synthesized by a novel, bacteriophage-specific mechanism.

Despite the fact that mature SP10c DNA contains dTMP, the acid-soluble fraction of infected cells contained no dTTP during the interval of phage replication. However, infected cells contained normal cellular levels of dATP, dGTP, and dCTP. Upon infection of deoxythymidine-starved Bacillus subtilis M160 (a deoxythymidine-requiring mutant of B. subtilis W23), mature phage DNA with a normal dTMP content was made. SP10c codes for an enzyme that seems to catalyze the tetrahydrofolate-dependent transfer of 1-carbon fragments to the 5 position of dUMP. The transfer of 1-carbon fragments is not accompanied by oxidation of tetrahydrofolage to dihydrofolate, implying that the enzyme in question is not a dTMP synthetase. It is proposed that dTMP in mature SP10c DNA is derived by the postreplicational modification of some other nucleotide and not by the direct incorporation of dTTP into DNA.     

噬菌体RB69 DNA聚合酶的表达、分离纯化和其聚合反应的稳态动力学研究

噬菌体ＲＢ６９外切酶活性缺失的ＤＮＡ 聚合酶突变体（Ｄ２２２Ａ／Ｄ３２７Ａ）在大肠杆菌细胞中表达,表达量达细胞蛋白总量的６９％ ．表达后经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦａｓｔＦｌｏｗ , Ｓｏｕｒｃｅ ３０Ｑ 和ＨＴＰ三步分离纯化,纯度可达９９％ 以上．随后测定了该酶利用５种ｄＮＴＰ为底物进行聚合反应的酶促动力学常数（Ｋｍ 和Ｋｃａｔ）,结果表明该酶利用ｄＵＴＰ的能力与利用ｄＴＴＰ的能力相近,Ｋｍ （ｄＴＴＰ）和Ｋｍ（ｄＵＴＰ）均较高于其它３种脱氧核苷酸的Ｋｍ （ｄＡＴＰ, ｄＣＴＰ, ｄＧＴＰ）,推测其Ｋｍ 值的差异主要来源于Ｔ／Ｕ 碱基本身,而并非全部由ＧＣ碱基配对与ＡＴ碱基配对之间的氢键作用力的强弱差别所决定．     

Deoxyuridine triphosphate pools after polyoma virus infection

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.     

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil.

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.     

Inhibitory activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine on the salmonid herpesviruses, Oncorhynchus masou virus (OMV) and Herpesvirus salmonis

The highly potent and selective anti-herpesvirus agent, (E)-5-(2-bromovinyl)-2'deoxyuridine (BVdU), was examined for its inhibitory effect on the salmonid herpesviruses Oncorhynchus masou virus (OMV) and Herpesvirus salmonis (H. salmonis). Minimum inhibitory concentrations (MIC) of BVdU for OMV and H. salmonis were 1.25 and 3.0 micrograms/ml, respectively; these values were equal to or higher than those obtained for acyclovir or cytarabine. OMV DNA polymerase activity was reduced in a dose-dependent fashion by BVdU 5'-triphosphate (BVdUTP) within the concentration range of 3 to 30 microM. However, BVdUTP could also be substituted for the natural substrate, TTP, in the OMV DNA polymerase assay. It is postulated that the inhibitory action of BVdU on the salmonid herpesviruses is more or less similar to that on other herpesviruses and resides with respect to the inhibition of the virus DNA polymerase activity as well as incorporation of BVdU into the viral DNA.