Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis.

The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.     

Hybrid selection of mRNA with biotinylated DNA

A novel, convenient, and highly efficient hybrid selection procedure is described. The method utilizes the polymerase chain reaction (PCR) in which one of two primers is biotinylated at the 5'-terminus. The concentration of the biotinylated primer is 100 times that of the other to synthesize biotinylated single-stranded DNA (asymmetric PCR). After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA.DNA-biotin onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The quantitative physical recovery of selected mRNA is about 70% with about 33% retention of biological activity.     

Monofunctional DNA-platinum(II) adducts block frequently DNA polymerases.

The question of whether monofunctional DNA platinum(II) adducts block synthesis of DNA by purified DNA polymerases of different types and origin has been investigated by comparing the time dependence of synthesis arrest and of DNA adduct formation. Activated salmon testis DNA is used as a suitable substrate for DNA synthesis allowing to probe inhibition by platinum(II) monoadducts for the variety of inherent template-primers. Reaction amplitudes are related to defined mixtures of dichloro and chloroaqua platinum(II) complexes. It is found that (i) all investigated DNA polymerases seem arrested (100% efficiency) at bifunctional DNA adducts. (ii) human DNA polymerase beta bypasses most of the monofunctional lesions of the three platinum(II) complexes investigated. (iii) Klenow fragment is blocked by monoadducts with increasing efficiency in the order cis-diamminechloroaquaplatinum(II) (0%) less than meso-[1,2-bis(2,6- dichloro-4-hydroxyphenyl)ethylenediamine] chloroaquaplatinum(II) (50%) less than trans-diamminechloro-aquaplatinum(II) (75%). (iv) Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, Physarum polycephalum DNA polymerase alpha, and calf thymus DNA polymerase alpha appear to be arrested by monoadducts. According to these examples, blocking efficiencies depend on the cis/trans-stereogeometry of fixation of the carrier ligands at platinum(II) residues, on the size/chemical nature of the platin(II) carrier ligand and on the type/origin of DNA polymerase.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Sites of termination of in vitro DNA synthesis on cis-diamminedichloroplatinum(II) treated single-stranded DNA: a comparison between E. coli DNA polymerase I and eucaryotic DNA polymerases alpha.

We have compared the capacity of the large fragment of E. coli DNA polymerase I and highly purified DNA polymerases alpha from either Drosophila melanogaster embryos or calf thymus to replicate single-stranded M13 mp10 DNA treated with the antitumoral drug cis-diamminedichloroplatinum(II) (cis-DDP). We report that: a) although prokaryotic and eukaryotic enzymes have different structural complexity and dissimilar in vivo functions, their synthesis was blocked in vitro at similar sites on cis-DDP treated DNA; b) this inhibition occurred not only at d(G)n sequences, as previously reported for E. coli DNA polymerase I, (Pinto & Lippard (1985) Proc. Natl. Acad. Sci. USA, 82, 4616-4619) but also at other sequences which may represent putative cis-DDP-DNA adducts.     

DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.     

Utilization in vitro of deoxyuridine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus.

DNA polymerases alpha and beta (EC 2.7.7.7.) from calf thymus could utilize dUTP as a substrate for DNA synthesis as well as DNA polymerase I of Escherichia coli. Deoxyuridylate was incorporated into DNA by replacing deoxythymidylate and supported the further elongation of DNA chains on activated DNA or on the intiated homopolymers, poly(dA) . (dT)10 and poly(rA) . (dT)10. The rate of the incorporation of deoxyuridylate into DNA varied from 50 to 160% of that of deoxythymidylate, depending on the nature of the template primers and the species of DNA polymerase used. The apparent Km values for dUTP were very similar to those for dTTP. Uracil DNA-glycosylase excised efficiently the uracil residues in products of DNA polymerase reactions with either activated calf thymus DNA or initiated homopolymers.     

DNA polymerase III from Saccharomyces cerevisiae. II. Inhibitor studies and comparison with DNA polymerases I and II

The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase. Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively. The mitochondrial enzyme was insensitive to the drug. N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM). Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM). The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM). With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases. Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting. Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III. The results show that DNA polymerase III is distinct from DNA polymerase I and II.     

(E)-5-(2-bromovinyl)-2'-deoxyuridine-5'-triphosphate as a DNA polymerase substrate.

Time course of incorporation and the effect of 5'-triphosphate of the selective antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (bv5dUTP) on the incorporation of dTTP and dATP into template-primers of different structure were studied in E. coli DNA polymerase I Klenow fragment enzyme-catalyzed reactions. bv5dUTP could substitute for dTTP depending on the structure of template-primer. E.g. into calf thymus DNA incorporation of bv5dUMP was around 80% of that of dTMP at 30 minutes of incubation. The analog has also inhibited dTMP incorporation, net DNA synthesis, however, was hardly affected. The substrate properties of the analog were studied with [2-14C]-labelled bv5dUTP.     

Stimulation of DNA polymerase alpha by hypergravity generated by centrifugal acceleration.

Gravity alteration is known to influence cell proliferation. Here we tested the effects of hypergravity on the action of DNA polymerase alpha, one of the DNA replication enzymes in eukaryotes. Hypergravity was produced by horizontal centrifugal acceleration with a hand-made rotator. The reaction rate of DNA polymerase alpha in centrifuge tubes increased along with the acceleration up to 4g, when a plateau was reached. In contrast, no stimulation was observed with primase, DNA polymerase epsilon, and the E. coli DNA polymerase I Klenow fragment. Kinetic analysis of DNA polymerase alpha reactions revealed that, under high gravity conditions, the K(m) value for template DNA decreased while the V(max) stayed constant. In contrast, the centrifugal acceleration did not affect the K(m) values for deoxyribonucleoside triphosphates. These results suggest that the hypergravity enhances the activity of DNA polymerase alpha by increasing the affinity of the enzyme for template DNA. Such enhancement was more prominent with a low concentration of DNA polymerase alpha under low ionic conditions.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

Inhibition of in vitro DNA chain elongation of 5-trifluoromethyl-2'-deoxyuridine residue in the template.

5-Trifluoromethyl-2'-deoxyuridine (CF3dUrd) is incorporated into the DNA of mammalian cells in culture. We have synthesized oligonucleotides that allows site specific introduction of CF3dUrd residue into synthetic DNA oligonucleotide. We described here the utilization of these oligonucleotides as template for in vitro DNA synthesis. When CF3dUrd residue located at an internucleotide site in the template, the chain elongation was partially arrested one nucleotide after or before the CF3dUrd residue of template using Escherichia coli polymerase I (Klenow fragment) or human polymerase alpha (pol alpha). These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.     

Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.     

Two DNA polymerases of Escherichia coli display distinct misinsertion specificities for 2-hydroxy-dATP during DNA synthesis

The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E. coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G. On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G. These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E. coli.