Koehl P, Valladier P, Lefèvre J F, Fuchs R P
Groupe de Cancérogénèse et de Mutagénèse Moléculaire et Structurale, IBMC du CNRS, Strasbourg, France.
Nucleic Acids Res. 1989 Dec 11;17(23):9531-41. doi: 10.1093/nar/17.23.9531.
The NarI restriction enzyme recognition site, G1G2CG3CC, has been identified as a hotspot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF) on the basis of a forward mutation assay in plasmid pBR322 in the bacterium Escherichia coli. AAF binds primarily to the C-8 position of guanine residues, and the three guanines of the NarI site are similarly reactive. Despite this similar chemical reactivity, only binding of AAF to the G3 residue causes the -2 frameshift mutations. To study the mechanisms underlying the specificity of the mutagenic processing further, we monitored the structural changes induced by a single AAF adduct within the NarI site by means of CD spectroscopy and thermal denaturation. The NarI sequence was studied as part of the 12-mer ACCGGCGCCACA. The purification and characterization of the three isomers having a single AAF adduct covalently bound to one of the three guanines of this 12 mer are described. The analysis of the melting profiles of the duplexes formed when these three isomers are annealed with the oligonucleotide of complementary sequence shows the same destabilizing effect of the AAF adduct on the three DNA helices. It is also shown, from the CD spectra, that modification of guanine G1 or G2 by AAF does not induce major changes in the helical structure of DNA. On the other hand, modification of guanine G3 induces a change in the CD signal that suggests the formation of a local left handed structure within the 12-mer duplex. These results show the polymorphic nature of the DNA structure in the vicinity of an AAF adduct.
基于在大肠杆菌中对质粒pBR322进行的正向突变试验，NarI限制酶识别位点G1G2CG3CC已被确定为N - 2 - 乙酰氨基芴（AAF）诱导-2移码突变的热点。AAF主要与鸟嘌呤残基的C - 8位结合，NarI位点的三个鸟嘌呤具有相似的反应活性。尽管化学反应活性相似，但只有AAF与G3残基结合才会导致-2移码突变。为了进一步研究诱变过程特异性的潜在机制，我们通过圆二色光谱（CD光谱）和热变性监测了NarI位点内单个AAF加合物诱导的结构变化。NarI序列作为12聚体ACCGGCGCCACA的一部分进行研究。本文描述了三种异构体的纯化和表征，这三种异构体分别将单个AAF加合物共价结合到该12聚体的三个鸟嘌呤之一上。当这三种异构体与互补序列的寡核苷酸退火形成双链体时，对其熔解曲线的分析表明，AAF加合物对三条DNA螺旋具有相同的去稳定作用。从CD光谱还可以看出，AAF对鸟嘌呤G1或G2的修饰不会引起DNA螺旋结构的重大变化。另一方面，鸟嘌呤G3的修饰会引起CD信号的变化，这表明在12聚体双链体内形成了局部左手结构。这些结果表明了AAF加合物附近DNA结构的多态性。
