Kenneth L. Beattie

Mutagenic consequences of the incorporation of 6-thioguanine into DNA

6-Thioguanine (S6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508-514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M13lacISaXb in reactions that omitted dGTP (-G) or dATP (-A). LacI mutation frequency was determined by beta-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducer). When a high concentration of SdGTP (24 microM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the -G and -A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the -G + S6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the -A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S6G with thymine leads to mutagenic effects in this assay.

Genetic relatedness of human DNA polymerase β and terminal deoxynucleotidyltransferase

The Protein Identification Resource (PIR) protein sequence data bank was searched for sequence similarity between known proteins and human DNA polymerase beta (Pol beta) or human terminal deoxynucleotidyltransferase (TdT). Pol beta and TdT were found to exhibit amino acid sequence similarity only with each other and not with any other of the 4750 entries in release 12.0 of the PIR data bank. Optimal amino acid sequence alignment of the entire 39-kDa Pol beta polypeptide with the C-terminal two thirds of TdT revealed 24% identical aa residues and 21% conservative aa substitutions. The Monte Carlo score of 12.6 for the entire aligned sequences indicates highly significant aa sequence homology. The hydropathicity profiles of the aligned aa sequences were remarkably similar throughout, suggesting structural similarity of the polypeptides. The most significant regions of homology are aa residues 39-224 and 311-333 of Pol beta vs. aa residues 191-374 and 484-506 of TdT. In addition, weaker homology was seen between a large portion of the nonessential N-terminal end of TdT (aa residues 33-130) and the first region of strong homology between the two proteins (aa residues 31-128 of Pol beta and aa residues 183-280 of TdT), suggestive of genetic duplication within the ancestral gene. On the basis of nucleotide differences between conserved regions of Pol beta and TdT genes (aligned according to optimally aligned aa sequences) it was estimated that Pol beta and TdT diverged on the order of 250 million years ago, corresponding roughly to a time before radiation of mammals and birds.

High-performance liquid chromatographic purification of deoxynucleoside 5′-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2-deoxynucleoside 5-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pure dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. epsilon dAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.

Utilization of 2′-Deoxy-6-thioguanosine 5′-Triphosphate In DNA Synthesis Catalyzed by DNA Polymerase I Klenow Fragment of Escherichia Coli

Abstract The substrate kinetics of 2′-deoxy-6-thioguanosine 5′-triphosphate for DNA polymerase I (Klenow fragment) of Escherichia coli were investigated. The analog nucleotide was readily incorporated in place of dGTP and it was misincorporated in place of dATP at low but detectable frequency.

Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli

Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking (minus reaction) or present at very low concentration (micro reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the -A reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a -A reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5-pyrimidine, 3-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts.

Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli

To test whether the identity of divalent metal activator affects the specificity of misincorporation during polymerization catalyzed by E. coli DNA polymerase I, we carried out the following procedure. A series of oligonucleotide primers, annealed at different positions along the lacZ region of bacteriophage M13mp9 DNA, were elongated in the presence of 3 of the 4 deoxynucleoside 5-triphosphates (dNTPs) until one or a few misincorporations occurred in each elongated primer. The elongated primers (containing deoxynucleotide residues that had been misincorporated in the presence of either Mg2+ or Mn2+) were then isolated and sequenced by the dideoxy chain termination method to determine the identity of deoxynucleoside monophosphates (dNMPs) that had been misincorporated at different template positions during the original minus reactions, activated by Mg2+ or Mn2+. The results obtained by this approach revealed that both the type of misincorporation and the effect of substituting Mn2+ for Mg2+ depended on the nucleotide sequence of the template. At 40% of the template positions at which misincorporation was compared with both metal ions (8 out of 20), the identity of mispairs differed significantly for synthesis activated by Mn2+ versus Mg2+. Of these 8 sites, 4 exhibited increased transversions in the presence of Mn2+, while 4 exhibited decreased transversions with Mn2+.

Genetic assay of misincorporation.

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3-terminus of the primer. Nearly all of the mutants induced by -A conditions were T----C base substitutions, and those induced by -G conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.

Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase β

Abstract A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Polβ and TdT to investigate the region of Polβ required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154–212 of TdT was replaced by the corresponding region encoding aa 1–60 of Polβ. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of Polβ represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Polβ protein possessed significant template-dependent polymerase activity suggests that aa 1–60 of Polβ are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Polβ possesses ssDNA-binding activity [Kumar ., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar ., Biochemistry 29 (1990b) 7156-7159; Prasad ., J. Biol. Chem. 268 (1993) 22746-22755].

In vitro mutagenesis in the lacI gene of Escherichia coli: Fate of 3′-terminal mispairs versus internal base mispairs in a transfection assay

The fate of G.T mismatches and frameshifts, present at the 3-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G.T mismatches in the middle of the duplex region or at the 3-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G.T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs (complete reaction) or in the absence of dATP (-A reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i-mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3-terminal G.T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G.T mismatches are degraded by the 3-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3-terminal mispairs. Single, double and triple T----C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the -A misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T----C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic minus conditions was due to the efficient action of the 3-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay.