Abstract
O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%. These frequencies were independent of the activity of the O6-alkylguanine DNA alkyltransferase. All three O4-alkylthymines induced T-->C transitions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic but strongly inhibited plasmid replication (> 90%). These results indicate that O4-alkylthymines are efficiently recognized by the nucleotide excision repair system and cause a complete cessation of plasmid replication if this system is deficient. Nevertheless, proficiency in the nucleotide excision repair pathway correlates with a high frequency of mutation induction by these lesions.
Highlights
04-Alkylthymines have been implicated as potential was obtained that the miscoding potential of these residues carcinogenic DNAlesionsW . e have studied the effecotfs might be very high [4,5,6]
But strongly inhibited plasmid replicat(i>ogno%). These lesions are substratesfor the ada- and ogt-encoded alkyltransresultsindicatethat04-alkylthyminesareefficiently ferases that normally act on 06-alkyldeoxyguanosine residues recognizedby the nucleotide excision repair system ainndDNA [9, 14, 15]. 04-EtT can be removed by nucleotide cause a complete cessation of plasmid replication if theixscision repair if alkyltransferases arenot inducedand there is systemisdeficient.,proficiencyinthe virtually no repair of these lesions in bacterial cells that lack nucleotide excision repair pathway correlates with a both the alkyltransferases and the nucleotide excision repair high frequency of mutation induction by these lesions. system [16]
We investigated whether 04-AlkTs in costs of publication of this article were defrayed in part by the payment human cells are substrate for nucleotide excision repair by of page charges
Summary
With ethanol, and the gapped duplexes were separated from the linear Analysis of Site-specific Modified Plasmids-Constructs with starting material by electrophoresis on agarose gels and subsequent an 04-AlkT-,an 06-MedG, or both an 04-EtTplus dG-C8-AAF'. Multiple mutations were found with pair proficient cell lines was approximately haloff that induced These frequencies werealso determined for undamaged (Wt) plasmids. Derived from pSVctr control plasmids (Fig. 4) In both of the excision repair-proficient cell lines, adducted possible influence of the alkyltransferase on the mutagenicity constructs andcontrols carrying anunmodified oligonucleotide of 04-AlkT,we transfected plasmids carrying a single 06-MedG yielded approximately equal amounts of replicated plasmids at the ClaI site into the sacemllelines. 04-AlkTlesions wereless well replicated relative toboth of the hardly exceeded background levels (2%).The majority of the controls (undamaged pSVsupF and cotransfected pSVctr); the mutations consisted of a G + A transition at the former posiamount of progeny obtained from adducted constructs waosnly tion of the adducted dG (Table V), which is in good agree-.
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