In vitroDNA replication implicates O²-ethyldeoxythymidine in transversion mutagenesis by ethylating agents

Abstract

A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.

A rodent model for Wilms tumors: embryonal kidney neoplasms induced by N-nitroso-N'-methylurea.

“…This mutation is identical to the one discovered in the WTI gene from a human leukemia cell line, CEM (2)(3)(4) (35)(36)(37). The premutagenic lesion 02-dT, a DNA adduct formed by N-ethyl-N'-nitrosourea, a compound closely related to NMU, is among the least efficiently repaired and, therefore, the most persistent in mammalian cells (36,37). Incorporation of dA and dT occurs opposite the premutagenic lesion.…”

“…Incorporation of dA and dT occurs opposite the premutagenic lesion. Whereas incorporation of dA impedes DNA synthesis, incorporation of dT is efficiently extended, leading to ART --TEA transversion (35)(36)(37). In the remaining three tumors, the C --T transitions concur with the well-known mutagenic activity of NMU, the formation of 06-methylguanine adducts (35)(36)(37) in the nontranscribed strand of DNA.…”

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

“…Correction for the slightly lower proportion of A-T base pairs in the lacZ gene results in a revised estimate of 73% mutations in A-T base pairs, which would not affect these comparisons. The most prominent classes of mutations induced by ENU in this study (Table 3) are (i) AT -* TA transversions, presunmably induced by 02-ethylthymine (32), (ii) G-C --AT transitions in non-5'-CpG sites, thought to be caused by 06-ethylguanine adducts (27), and (iii) A-T --G-C transitions, reported to be caused by 04-ethylthymine adducts (33). Mutant frequency is a function of a number of interrelated variables including the level of adduct deposition, DNA repair, and DNA replication (13).…”

Functional Nucleotide Excision Repair Is Required for the Preferential Removal of N-Ethylpurines from the Transcribed Strand of the Dihydrofolate Reductase Gene of Chinese Hamster Ovary Cells

“…Confluent CHO B11 cells were subcultured at a 1:10 dilution for experimental purposes. Following overnight incubation to allow for reattachment, the cells were grown in medium supplemented with 10 M [methyl- 3 H]thymidine (0.2 Ci/ml) for 24 h to radiolabel cellular DNA. Exponentially growing cells were exposed to ENU dissolved directly in medium in which the dialyzed fetal bovine serum was replaced with sterile water.…”

“…ENU reacts with nitrogen or oxygen atoms of purines, pyrimidines, and phosphodiester groups, resulting in an array of lesions in DNA (52,53). The modified DNA bases formed include the stable moieties O 6 -ethylguanine, O 4 -ethylthymine, O 2 -ethylthymine, and O 2 -ethylcytosine; all of these have been shown to cause base misincorporation during replication (2,3,18,57). A number of inherently unstable N-ethylpurines, including 7-ethylguanine (7-EtG) and 3-ethyladenine (3-EtA), are formed as well.…”