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.     

Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis.

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.     

Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases

Repair replication of short synthetic DNA's corresponding to parts of the gene for the major yeast alanine transfer RNA has been studied. The enzymes used were the Escherichia coli, Microccua luteus and T4 DNA polymerases, similar results being obtained with all the three enzymes. The DNA's used (Fig. 1) were: four double-stranded DNA's with the termini containing the 5′-hydroxyl group protruding by one, three, four or ten nucleotide units and two single-stranded DNA's 29 units long which are capable of folding back on themselves. The aspects inve tigated were: (1) completion of repair, (2) the minimum size of the polynucleotide chains required as primers and those which can serve as templates and (3) the minimum size of the primers which can abolish hairpin formation so as to give the “normal” repair replication. Repair replication of DNA's (DNA-I to DNA-IV: Fig. 1) was characterized to be essentially complete. The minimum size of the primer for repair replication of an extended single-stranded deoxypolynucleotide was ascertained to be about five to seven units long, while the primers required to overcome the hairpin formation in DNA-V and DNA-VI were found to be about twelve units long. Thus, in the presence of a suitable primer, the nucleotide incorporation which occurs in DNA-V in the absence of added primer was completely replaced by incorporation expected for normal repair replication. The minimum size of a chain which can serve as a template was also found in the present work to be about 12 units long.     

Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli

The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.     

Studies on the biochemical basis of spontaneous mutation. II. The incorporation of a base and its analogue into DNA by wild-type, mutator and antimutator DNA polymerases

The incorporation and turnover of adenine and its analogue 2-aminopurine into DNA by purified wild-type, mutator, and antimutator T4 DNA polymerase have been measured. Antimutators incorporate less 2-aminopurine into DNA than does wild type, and imitators incorporate more than wild type. Analysis of these data is consistent with the idea that the incorporation of 2-aminopurine is influenced primarily by the ratio of 3′-exonuclease to polymerase activities of the different enzymes. The experimental results conform to a model equation, which expresses the base incorporation frequency as a function of the polymerase insertion and removal activities. Some of the implications of the model equation are examined in this and the accompanying paper.     

Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.     

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.     

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.     

Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.