Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents.
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.
Op het Veld
C W
CW
MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, The Netherlands.
van Hees-Stuivenberg
S
S
van Zeeland
A A
AA
Jansen
J G
JG
D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004260:DNA Repair; D005020:Ethyl Methanesulfonate / Q000633:toxicity*; D005038:Ethylnitrosourea / Q000633:toxicity*; D005796:Genes / Q000187:drug effects*; D006147:Guanine / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000187:drug effects* / Q000235:genetics*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D013045:Species Specificity; D013941:Thymine / Q000378:metabolism
D000477:Alkylating Agents; D009153:Mutagens; D006147:Guanine; D004247:DNA; D005020:Ethyl Methanesulfonate; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea; D013941:Thymine