In vivo repair of ENU-induced oxygen alkylation damage by the nucleotide excision repair mechanism in Drosophila melanog
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O R I GI N A L P A P E R
L. Tosal á M. A. Comendador á L. M. Sierra
In vivo repair of ENU-induced oxygen alkylation damage by the nucleotide excision repair mechanism in Drosophila melanogaster Received: 26 October 1999 / Accepted: 20 November 2000 / Published online: 26 January 2001 Ó Springer-Verlag 2001
Abstract DNA damage caused by oxygen alkylation of bases (mainly at O6-G, O4-T and O2-T positions in DNA) has been correlated with the mutagenic and carcinogenic potency of monofunctional alkylating agents. In all kinds of organisms, repair of O6-alkylG is carried out mainly by the enzyme O6-methyl guanine-DNA methyltransferase (MGMT). However, little is known about the repair of the O-alkylT adducts or about the contribution of nucleotide excision repair (NER) to this process, especially in higher eukaryotes. To study the in¯uence of the NER system on the repair of O-alkylation damage, the molecular mutation spectrum induced by N-ethyl-N-nitrosourea (ENU) in an NER-de®cient Drosophila strain, carrying a mutation at the mus201 locus, was obtained and compared with a previously published spectrum for NER-pro®cient conditions. This comparison reveals a clear increase in the frequency of base pair changes, including GC ® AT and AT ® GC transitions and AT ® TA transversions. In addition, one deletion and two frameshift mutations, not found under NER-pro®cient conditions, were isolated in the NER-de®cient mutant. The results demonstrate that: (1) N-alkylation damage contributes considerably (more than 20%) to the mutagenic activity of ENU under NER-de®cient conditions, con®rming that the NER system repairs this kind of damage; and (2) that in germ cells of Drosophila in vivo, NER seems to repair O6-ethylguanine and/or O2-ethylcytosine, O4-ethylthymine, and possibly also O2-ethylthymine.
Communicated by D. Gubb L. Tosal á M. A. Comendador á L. M. Sierra (&) Departamento de BiologõÂ a Funcional e Instituto Universitario de OncologõÂ a del Principado de Asturias, AÂrea de GeneÂtica, c/ JuliaÂn ClaverõÂ a s/n, Universidad de Oviedo, 33006 Oviedo, Spain E-mail: [email protected] Tel.: +34-985103889 Fax: +34-985103534
Key words Nucleotide excision repair á N-ethyl-N-nitrosourea (ENU) á O-alkylation damage á mus201 á Drosophila melanogaster
Introduction Alkylation of oxygen and nitrogen atoms in DNA is one of the most important actions of monofunctional alkylating agents (Singer 1976). Nitrogen alkylation, in general, is quantitatively more signi®cant than oxygen alkylation (Beranek 1990), and correlates with induction of chromosomal aberrations in higher eukaryotic organisms (Vogel and Natarajan 1982). In addition, studies on site-speci®c mutagenesis have revealed that transversions, frameshift mutations and small deletions may also result from nitrogen alkylation, either directly or through the induction of abasic sites (Sahill et al. 1985; Loeb and Preston 1986). This N-alkylation damage is repaired by the nucleotide excision repair mechanism (NER; Selby and Sancar 1990; Bronstein et al. 1992; Huang et al. 1
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