title: Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.
abstract: S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.
authors.affiliation: Hubrecht Institute and Cancer Genomics Center, 3584 CT Utrecht, The Netherlands.
authors.forename: Harma
authors.initials: H
authors.lastname: Feitsma
authors.forename: Ewart
authors.initials: E
authors.lastname: de Bruijn
authors.forename: Jose
authors.initials: J
authors.lastname: van de Belt
authors.forename: Isaac J
authors.initials: IJ
authors.lastname: Nijman
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D053843:DNA Mismatch Repair / Q000187:drug effects; D049914:DNA Repair-Deficiency Disorders / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004625:Embryo, Nonmammalian; D005038:Ethylnitrosourea / Q000633:toxicity*; D005306:Fertilization / Q000235:genetics; D005787:Gene Frequency; D005854:Germ Cells / Q000187:drug effects*; D018095:Germ-Line Mutation; D008297:Male; D016296:Mutagenesis / Q000187:drug effects* / Q000235:genetics; D009153:Mutagens / Q000633:toxicity; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D009153:Mutagens; D005038:Ethylnitrosourea