Paalman S R, Sung C, Clarke N D
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 1997 Sep 16;36(37):11118-24. doi: 10.1021/bi970740t.
DNA repair methyltransferases (MTases) are stoichiometric acceptor molecules that are irreversibly inactivated in the course of removing a methyl group from O6-methylguanine (meG)-DNA or O4-methylthymine (meT)-DNA. A new assay has been developed to determine the relative efficiency of repair of meG and meT. The assay is based on the deprotection of methylated restriction sites in synthetic oligonucleotides and can be used to measure meG repair or meT repair directly. More importantly, relative repair efficiencies can be measured in competition experiments, using each of the methylated oligomers in turn as an inhibitor of repair for the other. Relative repair rates are determined by numerical solution of the coupled rate equations that describe this competition to the experimental data. We find that the human MTase repairs meT about 35-fold less well than meG, qualitatively similar to earlier studies. Contrary to previous reports, however, we find that Escherichia coli Ada repairs meG and meT with nearly equal efficiency. This finding, in conjunction with other recent reports, may indicate that low meT repair is a relatively unusual characteristic of the human homolog.
DNA修复甲基转移酶（MTases）是化学计量的受体分子，在从O6-甲基鸟嘌呤（meG）-DNA或O4-甲基胸腺嘧啶（meT）-DNA上去除甲基的过程中会不可逆地失活。已开发出一种新方法来测定meG和meT的相对修复效率。该方法基于合成寡核苷酸中甲基化限制位点的去保护，可直接用于测量meG修复或meT修复。更重要的是，在竞争实验中可以测量相对修复效率，依次使用每种甲基化寡聚物作为另一种修复的抑制剂。相对修复率通过将描述这种竞争的耦合速率方程数值求解以拟合实验数据来确定。我们发现人类MTase修复meT的效率比修复meG低约35倍，这在定性上与早期研究相似。然而，与之前的报道相反，我们发现大肠杆菌Ada修复meG和meT的效率几乎相同。这一发现与其他近期报道一起，可能表明低meT修复是人类同源物相对不寻常的特征。
