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K Bebenek

Publications and source records attributed to K Bebenek.

46 records · Page 3Linked to original sources
PubMed1990

Frameshift errors initiated by nucleotide misincorporation.

Studies presented here on the fidelity of DNA synthesis in vitro support the hypothesis that a classical base-substitution intermediate (i.e., a misincorporated nucleotide) can yield a frameshift mutation. By using a fidelity assay specifically designed to detect minus-one-base errors, nucleotide substrate pool imbalances that have previously been shown to increase the rate of misincorporation are now shown to also increase minus-one-base frameshift error rates. Examination of the specificity of the errors produced in reactions with various dNTP pool imbalances and various DNA templates revealed that template nucleotides were preferentially lost when they had as a 5' neighbor a nucleotide complementary to the dNTP provided in excess. This suggests that when a misincorporated nucleotide is complementary to the next nucleotide in the template, a misaligned intermediate containing a correct terminal base pair can form and be extended by a DNA polymerase, leading to a frameshift mutation. We present evidence that the proposed mechanism may operate in vivo and discuss the implications of this model for frameshift mutations induced by DNA damage.

Base Composition↗
PubMed1989

Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase.

DNA-dependent DNA synthesis in vitro by human immunodeficiency virus-1 reverse transcriptase is relatively error-prone. The enzyme, whether recombinant or from virus particles, produces errors while replicating M13mp2 DNA at a rate that, if operative in vivo, would produce about five mutations per genome per round of replication. Sequence analysis of mutants resulting from in vitro synthesis demonstrates that errors are nonrandomly distributed. Base substitution and one-base frameshift mutational hot spots are observed. The specificity and position of errors suggest that most of the frameshifts and many of the base substitutions are initiated by template-primer slippage. Processivity analysis for the enzyme on the M13mp2 DNA template reveals strong termination at specific sites. Termination sites within homopolymer sequences correlate with frameshift mutational hot spots. The results suggest that the formation and/or utilization of misaligned template-primers is increased during the dissociation-reinitiation phase of the reaction.

Base Sequence↗
PubMed1989

Influence of dam and mismatch repair system on mutagenic and SOS-inducing activity of methyl methanesulfonate in Escherichia coli.

In contrast to earlier reports (Mohn et al., 1980; Glickman, 1982), we show that E. coli dam- cells are able to mutate following MMS treatment. Since the mutagenicity of MMS has been regarded as largely dependent on induction of the SOS functions, E. coli strains bearing the recA::lacZ or umuC::lacZ fusions were used to determine the ability of MMS to induce the SOS functions in the various dam+ and dam- strains. The mutagenicity of MMS was also tested in several of these strains. The results show that (i) there is no direct correlation between SOS-inducing ability and mutagenicity potency of MMS; and (ii) most of the premutagenic lesions induced by MMS are removed from DNA of dam+ or dam- cells by the mismatch repair system. The role of strand breaks in repair of mismatches induced by alkylating agents is discussed.

DNA Repair↗
PubMed1989

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins.

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.

Animals↗
PubMed1988

The accuracy of reverse transcriptase from HIV-1.

A study was conducted to determine the fidelity of DNA synthesis catalyzed in vitro by the reverse transcriptase from a human immunodeficiency virus type 1 (HIV-1). Like other retroviral reverse transcriptases, the HIV-1 enzyme does not correct errors by exonucleolytic proofreading. Measurements with M13mp2-based fidelity assays indicated that the HIV-1 enzyme, isolated either from virus particles or from Escherichia coli cells infected with a plasmid expressing the cloned gene, was exceptionally inaccurate, having an average error rate per detectable nucleotide incorporated of 1/1700. It was, in fact, the least accurate reverse transcriptase described to date, one-tenth as accurate as the polymerases isolated from avian myeloblastosis or murine leukemia viruses, which have average error rates of approximately 1/17,000 and approximately 1/30,000, respectively. DNA sequence analyses of mutations generated by HIV-1 polymerase showed that base substitution, addition, and deletion errors were all produced. Certain template positions were mutational hotspots where the error rate could be as high as 1 per 70 polymerized nucleotides. The data are consistent with the notion that the exceptional diversity of the HIV-1 genome results from error-prone reverse transcription.

Avian Myeloblastosis Virus↗
PubMed1987

Are Escherichia coli dam- as compared to dam+ hypermutable by base analogs?

It was shown that some base analogs, like n2Pur and n2oh6Ade (but not n2om6Ade or oh4Cyd) which strongly inhibit growth of dam- cells, mutagenize and preselect dam- populations. As a result dam- mut(-)--devoid of mismatch repair, dam+ revertants, or dam(-)--insensitive to a base analog are exclusively obtained after mutagenesis. The composition of these mutants depends on the base analog applied. By using reconstruction experiments, the frequency of these mutations induced by n2Pur was calculated.

DNA Mutational Analysis↗
PubMed1985

Ability of base analogs to induce the SOS response: effect of a dam mutation and mismatch repair system.

2-Aminopurine, 2-amino-N6-hydroxyadenine, 2-amino-N6-methoxyadenine and 2-amino-N6-methyl-N6-hydroxyadenine (but not N4-hydroxycytidine), strong mutagens of base analog type, may induce the SOS response in E. coli cells. This ability is greatly enhanced in dam3 mutants and abolished in dam3mutS, dam3mutH, and dam3mutL strains, thereby suggesting that the mismatch repair system is involved in the mechanism of induction.

DNA Repair↗
PubMed1983

Involvement of the mismatch repair system in base analogue-induced mutagenesis.

The influence of the mismatch repair system, and the role of mutS, mutR, and mutL genes, in mutagenesis induced by n2ome6Ade, n2oh6Ade and n2Pur have been investigated. From the frequency of reversion of Arg-Thr- and His- markers in AB2497, and its mut- derivatives, it was concluded that mismatches introduced by n2-ome6Ade and n2oh6Ade are better substrates for mismatch repair enzymes than that introduced by n2Pur. All these mut-gene products are more active in removing spontaneous or base analogue-induced mismatches which, when unexcised, lead to transversion of base repairs, than those which lead to transitions. Active engagements of mutL, mutR, or mutS gene products depend on the kind of mutation, the site of mutagenesis, and the inducing agent. Dam- cells are over-mutated by both n2ome6Ade and n2oh6Ade, but are hyper-sensitive to n2oh6Ade only. It is proposed that hyper-sensitivity of dam- cells is due not only to an increase in overlaping gap formation on both strands of DNA, but to a greater lability of the impaired cells. Results are presented which strongly suggest that n2ome6Ade in mut+ cells and n2oh6Ade in mut- only, can induce GC leads to TA transversions.

2-Aminopurine↗