Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line
Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line
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DOI：
10.1093/mutage/gen019
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发表时间：
2008-07-01
期刊：
影响因子：
2.7
通讯作者：
Cuppen, Edwin
中科院分区：
文献类型：
--
作者：
Feitsma, Harma;de Bruijn, Ewart;Cuppen, Edwin
S（N）1型烷化剂如N-乙基-N-亚硝基脲（ENU）是非常有效的诱变剂。它们的作用是将烷基转移到DNA碱基上，一旦复制过程中出现错配，可能会在下一个复制周期中引起单碱基对突变。由于DNA错配修复（MMR）蛋白参与识别烷基化损伤，我们假设在MMR缺陷的背景下，ENU诱导的突变率可能会增加，这将有利于诱变方法。我们应用标准ENU诱变方案，成年斑马鱼MMR基因msh 6和杂合对照缺陷，以研究MMR对ENU诱导的DNA损伤的影响。纯合和杂合突变体的剂量依赖性致死率相似，表明ENU抗性无差异。通过对诱变鱼的异交后代中的基因组靶点进行高通量双脱氧重测序发现突变，也没有发现生殖系突变频率的任何差异。这些结果可能表明，斑马鱼的最大突变负荷已达到目前使用的高度优化的ENU诱变方案。或者，斑马鱼生殖系中的MMR系统可能非常迅速地饱和，从而对高剂量ENU诱变具有有限的作用。
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.