Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.
Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.
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O6-甲基鸟嘌呤切口：人体细胞提取物导致 DNA 中胸腺嘧啶错配。
DOI：
10.1021/bi00149a034
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Day3rd,RS
中科院分区：
文献类型：
--
作者：
Sibghat-Ullah;Day3rd,RS
1992年4月8日收到的修订手稿摘要：人类无细胞提取物用于检测特异性切割DNA中与C或T配对的C^-甲基鸟嘌呤（m6 G）的活性。一个45 bp的双链DNA含有一个m6 G从一个T（m6 G：T）是测试底物。胶质母细胞瘤细胞系A172和A1235（缺乏m6 G特异性修复蛋白m6 G-DNA甲基转移酶，MGMT）和结肠癌细胞系HT 29（含有MGMT）的提取物显示出对m6 G：T [和G：T，如Wiebauer和Jiricny（1989）先前报道的]底物的T链的特异性切割活性，但不切割m6 G：C（或G：C）底物。竞争实验表明，这种活性与人类细胞切割G：T错配的活性相似，如果不是完全相同的话。切割位点与人G：T或G：A特异性错配酶识别的位点相似，即磷酸二酯键在3 '和5'端与匹配不良的T结合，表明糖酵解去除匹配不良的T，然后通过I类或II类AP核酸内切酶进行主链切割。MGMT失活的三个实验表明，m6 G：T切割活性不仅仅是由于两步机制，其中MGMT将首先介导m6 G：T底物转化为G：T底物，G：T底物将用作G：T切割的底物。HT 29的提取物含有一种DNA结合因子，可能是DNA序列特异性的，它抑制m6 G：T（而不是G：T）底物的切割，该底物通过向反应中加入合成DNA而被去除。当DNA鸟嘌呤残基与SN 1甲基化剂如MNNG和MNU反应时，在DNA中产生m6 G（Lawley，1976），并在正常细胞中由22-kDa蛋白MGMT修复，该蛋白MGMT将m6 G甲基转移到特定的MGMT残基，从而恢复鸟嘌呤的正确结构（Olsson & Lindahl，1980; Harris埃塔尔，1983; Yarosh等人，1983; Foote等人，1983年）。约20-30%的人实体瘤细胞系显示Mer-表型（Day等人，1980 a），lack MGMT [Yarosh埃塔尔. (1983，1984）和Sibghat-Ullah等人，未证实的结果]，并且如增强的致死率所示对MNNG超敏感（Day等人，
Revised Manuscript Received April 8, 1992 abstract: Human cell-free extracts were used to detect activities specifically incising C^-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G: T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell lineHT29, containing MGMT, showed incisionactivities specific for the T strand of m6G: T [and G: T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G: C (or G: C) substrates. Competition experimentsshowed that the activity was similar to, if not identical with, the activity in human cells that incises G: T mismatches. The incision sites were similar to those recognized by human G: T-or G: A-specific mismatch enzymes, ie, the phosphodiester bonds both 3'and 5'to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G: T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G: T substrate to a G: T substrate which would serve as a substrate for G: T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G: T (but not the G: T) substrate, that was removed by the addition of synthetic DNA to the reaction.We are interested in understanding the biological effects produced by unrepaired C^-methylguanine (m6G)'in DNA. m6G is produced in DNA when DNA guanine residues react with such SN1methylating agents as MNNG and MNU (Lawley, 1976) and is repairedin normal cells by the 22-kDa protein MGMT that transfersthe m6G methyl group to a specific MGMT residue, thus restoring proper structure to guanine (Olsson & Lindahl, 1980; Harris etal., 1983; Yarosh et al., 1983; Foote et al., 1983). About 20-30% of human solid tumor cell lines show the Mer-phenotype (Day et al., 1980a), lack MGMT [Yarosh etal.(1983, 1984) andSibghat-Ullah et al., unpublishedresults], and are hypersensitive to MNNG as indicated by enhanced lethality (Day et al.,