Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents

Abstract

The role of the nucleotide excision repair (NER) pathway in removal of DNA ethylation damage was investigated by means of hprt mutational spectra analysis in the NER-deficient Chinese hamster ovary cell line UV5, which lacks ERCC2/XPD, and in its parental cell line AA8. Both cell lines were exposed to ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gave a similar dose-dependent increase in hprt mutants in UV5 compared with AA8. In both cell lines EMS-induced mutations in the hprt coding region consisted almost exclusively of GC-->AT transitions, probably due to the direct miscoding lesion O6-ethylguanine. ENU, an agent that in addition to O6-ethylguanine also induces other O-alkylation products, was significantly more mutagenic in UV5 than in AA8. Mutational spectra analysis showed that the proportions of ENU-induced GC-->AT, AT-->TA and AT-->GC base pair changes were similar for both cell lines. ENU-induced DNA lesions that may be involved in GC-->AT transitions are O6-ethylguanine and O2-ethylcytosine, the latter being a chemically stable DNA lesion of which the miscoding properties and repair characteristics are largely unknown. ENU-induced AT-->TA transversions are probably caused by O2-ethylthymine, which mispairs with thymine. In AA8 thymines in ENU-induced AT-->TA transversions were exclusively located in the non-transcribed strand of the hprt gene, whereas in UV5 30% of these thymines were found in the transcribed strand. Together, these results indicate that O6-ethylguanine is a poor substrate for NER in rodent cells and that O2-ethylpyrimidines are preferentially removed from the transcribed strand of the hprt gene by NER.

Direct, genome-wide assessment of DNA mutations in single cells

“…Analysis of the ENU-induced mutations falling within genic regions in the two MEF cells showed evidence of transcription-coupled repair, with a lower fraction of ENU-induced mutations occurring at T and G bases, the predominant adduct bases, on the transcribed strand than the non-transcribed strand ( P  < 0.01) (Figure 3B). This bias appeared strongest for TA transversions ( P  < 0.0002), supporting previous results at the endogenous HPRT gene locus (22). No evidence for any transcription-coupled repair process (Figure 3C) was seen in the S2 cells, in keeping with results obtained with Drosophila Kc cells (23) and the absence of homologues of either CSA or CSB, the main TCR genes, in Drosophila (24).…”

“…In spite of this difference, the similarity between the two species also at this level is striking. The spontaneous mutation spectra observed in the untreated cells showed considerably lower levels of A:T →T:A mutations, which are known to be highly enriched following treatment with alkylating agents (12,22).

Figure 3.Somatic point mutation spectra and localization.

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Role of Nicotinamide in DNA Damage, Mutagenesis, and DNA Repair

“…Some chemotherapeutic agents (e.g., 5-fluorouracil, 6-mercaptopurine) also interfere with tryptophan conversion to niacin [36]. Moreover, chemotherapeutic alkylating agents have been shown to cause miscoding lesions, chromosomal aberrations [37], and secondary cancer, particularly leukemia, which complicates chemotherapy in 10%–15% of cancer survivals [38]. More direct evidence comes from studies in rats, which showed that niacin deficiency significantly increases the risk of chemotherapeutic-induced secondary leukemia [39].…”

Single-cell transcriptogenomics reveals transcriptional exclusion of ENU-mutated alleles

“…In comparison with gSNVs (Supplementary Table 5) or spontaneous somatic mutations [8], the mutation spectrum of sSNVs exhibited a significantly higher rate of AT:TA transversions, the signature mutations of ENU [36, 37]. Furthermore, in comparison with the sSNVs, the gSNVs detected in exons comprised no nonsense variants and a significantly lower ratio of mis-sense to silent variants (Supplementary Table 6, p = 0.049, t-test), which is to be expected because of negative selection.…”

“…Allelic avoidance of somatic SNVs was observed for ENU-lesions located both in the transcribed and in the nontranscribed strands (Supplementary Table 7B). The slightly higher number of mutations occurring at nontranscribed strands most likely reflects the operation of transcription-coupled repair [8, 36]. The low density of ENU-induced mutations also rules out the possibility that mutant alleles are not transcribed due to a concomitant mutation in the promoter region of the same allele.…”