Mutagenesis by O6-methyl-, O6-ethyl-, and O6-benzylguanine and O4-methylthymine in human cells:: Effects of O6-alkylguanine-DNA alkyltransferase and mismatch repair
Mutagenesis by O6-methyl-, O6-ethyl-, and O6-benzylguanine and O4-methylthymine in human cells:: Effects of O6-alkylguanine-DNA alkyltransferase and mismatch repair
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DOI：
10.1021/tx010032f
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发表时间：
2001-07-01
影响因子：
4.1
通讯作者：
Moschel, RC
中科院分区：
文献类型：
--
作者：
Pauly, GT;Moschel, RC
当O-6-甲基（m（6）G）-、O-6-乙基（e（6）G）-和O-6-苄基鸟嘌呤（B（6）G）以及O-4-甲基胸腺嘧啶（m（4）T）位点特异性地掺入lacZ '基因的ATG起始密码子中时，使用双链和带缺口穿梭载体来研究这些碱基在人细胞中的诱变。将载体转染入人肾细胞（293）或结肠肿瘤细胞（SO）或错配修复缺陷型人结肠肿瘤细胞（H6和LoVo）。在转染前，通过用O-6-苄基鸟嘌呤处理细胞，任选地使细胞O-6-烷基鸟嘌呤-DNA烷基转移酶（烷基转移酶）失活。在烷基转移酶感受态细胞中，所有修饰碱基的致突变性在缺口质粒中比在双链质粒中显著更高。烷基转移酶失活增加了双链和缺口质粒中三个O-6-取代鸟嘌呤的诱变，但不影响m（4）T诱变。在不存在烷基转移酶的情况下，在错配修复缺陷型H6和LoVo细胞中，双链载体中的m（6）G和较小程度的e（6）G的诱变高于SO或293细胞，表明e（6）G和m（6）G在这些细胞中进行错配修复加工。m（4）T和B（6）G的诱变水平不受错配修复状态的影响。当插入有缺口的质粒中且不存在烷基转移酶时，修饰碱基的致突变性顺序为m（4）T > e（6）G，m（6）G > B（6）G。O-6位取代的鸟嘌呤主要产生G ->A跃迁，而m（4）T主要产生T ->C跃迁。然而，在错配修复缺陷的LoVo细胞中，除了T-C转换外，m（4）T还产生了大量的T ->A颠换突变。
Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O-6-methyl (m(6)G)-, O-6-ethyl (e(6)G)-, and O-6-benzylguanine (b(6)G), and O-4-methylthymine (m(4)T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ ' gene. Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo). Cellular O-6-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O-6-benzylguanine prior to transfection. In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids. Alkyltransferase inactivation increased mutagenesis by the three O-6-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis. In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells. The level of mutagenesis by m(4)T and b(6)G was not affected by mismatch repair status. When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m(4)T > e(6)G congruent to m(6)G > b(6)G. The O-6-substituted guanines primarily produced G -->A transitions while m(4)T primarily produced T -->C transitions. However, m(4)T also produced a significant number of T -->A transversion mutations in addition to T-C transitions in mismatch repair deficient LoVo cells.