C8-guanine adduct-induced stabilization of a -1 frame shift intermediate in a nonrepetitive DNA sequence.
C8-guanine adduct-induced stabilization of a -1 frame shift intermediate in a nonrepetitive DNA sequence.
复制标题
C8-鸟嘌呤加合物诱导非重复 DNA 序列中 -1 移码中间体的稳定。
作者：
Nolan,SJ;McNulty,JM;Krishnasamy,R;McGregor,WG;Basu,AK
通过热熔融实验研究了致癌物质1-硝基芘形成的主要DNA加合物n-（脱氧鸟苷-8-酰基）-1-氨基芘引起框架移位突变的机理，其中加合物两侧分别有一个5 ‘和3 ’ c。与未修饰的13-聚物相比，加合物使双链失稳4 - 5 kcal/mol，并且ΔΔGvalue保持大致相同，无论与加合物相对的是哪个碱基。相比之下，删除加合物对面的碱基使双相稳定了近4千卡/摩尔。将相同序列背景下的加合物插入含有猿猴病毒40复制源的噬菌体M13 DNA中。构建的DNA模板用正常人成纤维细胞提取物在体外复制。在双向半保守复制后，加合物没有从后代DNA中移除，这表明它被细胞提取物绕过，而不是修复。当评估新复制的噬菌体在修饰G区域的突变时，大多数噬菌体在加合物位点含有G，表明无错误复制。检测到少量突变体（~ 2 × 10-3），所有突变体都含有靶向G·C碱基对缺失。这表明DNA中加合物的热力学稳定性与人类DNA聚合酶在复制绕过过程中发生的错误之间存在关系。
The mechanism of frame shift mutagenesis induced byN-(deoxyguanosin-8-yl)-1-aminopyrene, the major DNA adduct formed by the carcinogen 1-nitropyrene, was investigated by thermal melting studies of a 13-mer in which the adduct was flanked by a 5‘ and a 3‘ C. Compared to the unmodified 13-mer, the adduct destabilized the duplex by 4−5 kcal/mol, and the ΔΔGvalue remained approximately the same regardless of which base was placed opposite the adduct. In contrast, deletion of the base opposite the adduct stabilized the duplex by nearly 4 kcal/mol. The adduct in the same sequence context was inserted into a bacteriophage M13 DNA containing the simian virus 40 origin of replication. The constructed DNA template was replicated in vitro with extracts from normal human fibroblasts. The adduct was not removed from the progeny DNA following bidirectional semiconservative replication, which suggests that it had been bypassed, rather than repaired, by the cell extract. When newly replicated bacteriophage was evaluated for mutations in the region of the modified G, most contained a G at the adduct site, indicating error-free replication. A small number of mutants (∼2 × 10-3) were detected, all of which contained a targeted G·C base pair deletion. This suggests a relationship between the thermodynamic stability of the adduct in DNA and the errors that occurred during replicative bypass by the human DNA polymerases.