The Effect of Mismatch-Specific Thymine DNA Glycosylase (TDG) Deficiency on Spontaneous Mutation in Mice

在哺乳动物细胞中，胞嘧啶(5-甲基胞嘧啶)在CpG位点的甲基化被认为在基因表达、胚胎发生、基因组印记和X染色体失活中起着关键作用。5-甲基胞嘧啶也是已知的致突变物质，因为它被水解性脱除，并转化为胸腺氨酸，导致G；T错配形成。因此，5-甲基胞嘧啶的这种脱氨基反应可能是G：C到A：T碱基置换的主要原因。TDG(错配特异性T_胸腺嘧啶D_NA G__糖基酶)是CpG位点胸腺嘧啶与G错配的修复酶之一。为了研究TDG(-/-)ES细胞在体内的功能，我们利用基因打靶技术获得了TDG(-/-)ES细胞。Nick试验显示TDG(-/-)ES细胞的G-T错配糖基酶活性较低，但HPRT基因突变频率在TDG(+/+)和(-/-)ES细胞间无差异。这一结果表明TDG对ES细胞的突变诱导作用可能不强。我们还产生了TDG(+/-)小鼠。他们是有生育能力和正常生长的，然而，纯合子突变(-/-)小鼠在后11.5天内死亡。野生型与TDG的自发突变率无显著差异。对于(-/-)胚胎，我们发现正常胚胎发育所必需的去甲肾上腺素水平在TDG(-/-)胚胎中显著降低。此外，使用去甲肾上腺素可以部分挽救TDG(-/-)胚胎，使胚胎发育超过11.5DPC。这些结果表明，TDG(-/-)胚胎的致死性部分是由于去甲肾上腺素的减少，TDG对胚胎的发育是不可或缺的。

In mammalian cells, methylation of cytosine (5-methylcytosine) at CpG site is thought to play a pivotal role in control of gene expression, embryogenesis, genomic imprinting, and X chromosome inactivation. 5-methylcytosine is also known to be mutagenic because it is dearninated hydrolytically and converted to thyrnine resulting in G;T mismatch formation. Thus, this deamination of 5-methylcytosine is implied to be the main cause of G:C to A:T base substitution.TDG (mismatch-specific T__-hymine D__-NA G__-lycosylase) is one of the repair enzymes working in removal of thymine mispairing with G at CpG site. We generated TDG (-/-) ES cells by gene targeting in order to investigate its function in vivo. Although TDG (-/-) ES cells had little G-T mismatch glycosylase activity in nicking assay, there is no difference in mutant frequency at Hprt gene between TDG (+/+) and (-/-) ES cells. This result suggests that TDG might not have strong effects on mutation induction in ES cells.We have also generated TDG (+/-) mice. They are fertile and grow normally however, homozygous mutant (-/-) mice died before ll.5 days post coitum (dpc). Although there was no significant difference in spontaneous mutant frequency between wild type and TDG . (-/-) embryos, we found that the level of noradrenaline, necessary for normal embryogenesis, dramatically reduced in TDG (-/-) embryos. Furthermore, we could partially rescue TDG (-/-) embryos beyind 11.5 dpc by the administration of noradrenaline. These results suggest that the lethality of TDG (-/-) embryo is in parts due to noradrenaline reduction, and TDG is indispensable for embryonic development.