The main role of human thymine-DNA glycosylase is removal of thymine produced by deamination of 5-methylcytosine and not removal of ethenocytosine
The main role of human thymine-DNA glycosylase is removal of thymine produced by deamination of 5-methylcytosine and not removal of ethenocytosine
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DOI：
10.1074/jbc.m211084200
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发表时间：
2003-03-07
影响因子：
4.8
通讯作者：
Waters, TR
中科院分区：
文献类型：
--
作者：
Abu, M;Waters, TR
氯乙烯的甲基化产物与DNA中的胞嘧啶反应生成3，N-4-乙烯基胞嘧啶。最近的研究表明，乙烯胞嘧啶是通过碱基切除修复途径修复的，其中乙烯碱基被胸腺嘧啶-DNA糖基化酶去除。在这里，单周转动力学已被用来比较切除胸腺嘧啶-DNA糖基化酶与切除胸腺嘧啶的乙烯胞嘧啶。研究了侧翼序列对乙烯胞嘧啶切除的影响。这里研究的34-bp双链体分为三类。在CpG位点内与鸟嘌呤碱基配对的乙烯胞嘧啶（即CpG。C-是DNA的一个元件）是迄今为止最好的底物，其特异性常数（k（2）/K-d）为25.1 × 10（6）M-1 S-1。其次是含有TpG. C-、GpG. C-和CpG. T的DNA双链体。它们的特异性常数比CpG-1C-DNA小45-130倍。最差的底物是含有ApG-1C和TpG.T的DNA双链体，其特异性常数分别比CpG低1，600和7，400倍。含有乙烯胞嘧啶的DNA比含有G.T错配的DNA结合得更紧密。这可能是因为胸腺嘧啶-DNA糖基化酶可以从G. C-中翻转出乙烯胞嘧啶，这比它从G.T错配中翻转出胸腺嘧啶更容易。由于胸腺嘧啶-DNA糖基化酶对乙烯胞嘧啶的去除具有较大的特异性常数，因此有人认为其主要目的是处理乙烯胞嘧啶。然而，这些结果表明，胸腺嘧啶-DNA糖基化酶在切除胸腺嘧啶和乙烯胞嘧啶时对CpG位点具有很强的序列偏好，这表明胸腺嘧啶-DNA糖基化酶在体内的主要作用是去除由CpG位点处5-甲基胞嘧啶脱氨基产生的胸腺嘧啶。
Metabolites of vinyl chloride react with cytosine in DNA to form 3,N-4-ethenocytosine. Recent studies suggest that ethenocytosine is repaired by the base excision repair pathway with the ethenobase being removed by thymine-DNA glycosylase. Here single turnover kinetics have been used to compare the excision of ethenocytosine by thymine-DNA glycosylase with the excision of thymine. The effect of flanking DNA sequence on the excision of ethenocytosine was also investigated. The 34-bp duplexes studied here fall into three categories. Ethenocytosine base-paired with guanine within a CpG site (i.e. CpG .C-is an element of-DNA) was by far the best substrate having a specificity constant (k(2)/K-d) of 25.1 x 10(6) M-1 S-1. The next best substrates were DNA duplexes containing TpG.C-is an element of, GpG.C-is an element of, and CpG.T. These had specificity constants 45-130 times smaller than CpG -'C-DNA. The worst substrates were DNA duplexes containing ApG-'C and TpG.T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG.C-is an element of-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G.T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G.C-is an element of base pair more easily than it can flip out thymine from a G.T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.