Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos.
S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.
Hubrecht Institute, Royal Academy of Arts and Sciences & University Medical Centre Utrecht, Cancer Genomics Center, 3584 CT, Utrecht, The Netherlands.
Harma
H
Feitsma
Alper
A
Akay
Edwin
E
Cuppen
D000477:Alkylating Agents / Q000633:toxicity*; D000478:Alkylation; D000818:Animals; D016923:Cell Death; D043171:Chromosomal Instability; D002869:Chromosome Aberrations; D004249:DNA Damage; D053843:DNA Mismatch Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000187:drug effects; D005038:Ethylnitrosourea / Q000633:toxicity*; D017353:Gene Deletion; D008770:Methylnitrosourea / Q000633:toxicity*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D015027:Zebrafish / Q000196:embryology / Q000235:genetics
D000477:Alkylating Agents; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D009153:Mutagens; D008770:Methylnitrosourea; D005038:Ethylnitrosourea