Quillardet P, Boscus D, Touati E, Hofnung M
Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS Ura 1444, Institut Pasteur, 25 rue du Dr. Roux, 75724, Paris Cedex 15, France.
Mutat Res. 1998 Dec 3;422(2):237-45. doi: 10.1016/s0027-5107(98)00203-6.
DNA adducts that block replication, induced in vivo by the 5-nitrofuran derivative R7000 (7-methoxy-2-nitronaphtho[2, 1-b]-furan) were mapped, at nucleotide resolution, in a region of the lacI gene of Escherichia coli, using a reiterative primer extension assay [D. Chandrasekhar, B. Van Houten, High resolution mapping of UV-induced photoproducts in the Escherichia coli lacI gene: inefficient repair of the non-transcribed strand correlates with high mutation frequency, J. Mol. Biol., 1994, Vol. 238, pp. 319-332]. It was found that R7000 induced a broad spectrum of low frequency replication blocks rather than particular hot spots in a limited number of particular targets. Most of these replication blocks were observed at G nucleotides, and most of G nucleotides present in the DNA sequence, if not all, constituted a possible target for the chemical attack of the compound. In addition, a large part of replication blocks observed at A, C or T could also reflect a replication block at the 3' or 5' nucleotide flanking a guanosine-DNA adduct. Only a very small number of replication blocks could be observed at A, C or T nucleotides non-adjacent to a G. These results show that, guanosine-DNA adducts are the main DNA lesions that block replication induced by R7000 in E. coli and suggests a strong reactivity of the genotoxic species generated in vivo by R7000 with the G nucleotidic targets. From 26 R7000-induced mutations previously mapped in this region [E. Touati, E. Krin, P. Quillardet, M. Hofnung, 7-methoxy-2-nitronaphto[2,1-b]furan (R7000)-induced mutation spectrum in the lacI gene of Escherichia coli: influence of SOS mutagenesis, Carcinogenesis, 1996, Vol. 17, pp. 2543-2550.], 22 (85%) occurred at GC base pairs at which termination products were observed. The other mutagenic events involved AT base pairs adjacent to a G nucleotide forming a replication block. Thus all mutagenic events occurred at, or adjacent to, a G nucleotide forming a replication block. Although it could not be excluded that some mutagenic events are due to undetected DNA lesions that do not block replication, these results strongly suggest that guanosine-DNA adducts that block DNA replication are responsive for a large part of the mutagenic events generated by R7000. The powerful capacity of R7000 to form adducts at most of the guanosine residues in a DNA sequence may account for at least part of its very potent genotoxic properties.
利用重复引物延伸分析法，在核苷酸分辨率水平上，对由5-硝基呋喃衍生物R7000（7-甲氧基-2-硝基萘并[2,1-b]呋喃）在体内诱导产生的、阻断复制的DNA加合物，在大肠杆菌lacI基因的一个区域进行了定位[D.钱德拉塞卡尔，B.范霍滕，大肠杆菌lacI基因中紫外线诱导光产物的高分辨率定位：非转录链的低效修复与高突变频率相关，《分子生物学杂志》，1994年，第238卷，第319 - 332页]。研究发现，R7000诱导产生了广泛的低频复制阻断，而非在有限数量的特定靶点中的特定热点。这些复制阻断大多出现在G核苷酸处，并且DNA序列中存在的大多数G核苷酸，即便不是全部，都构成了该化合物化学攻击的可能靶点。此外，在A、C或T处观察到的大部分复制阻断，也可能反映了鸟苷 - DNA加合物侧翼3'或5'核苷酸处的复制阻断。在与G不相邻的A、C或T核苷酸处，仅能观察到极少数的复制阻断。这些结果表明，鸟苷 - DNA加合物是R7000在大肠杆菌中诱导阻断复制的主要DNA损伤，并表明R7000在体内产生的遗传毒性物质与G核苷酸靶点具有很强的反应活性。从先前在该区域定位的26个R7000诱导的突变[E.图阿蒂，E.克林，P.基利亚尔代，M.霍夫nung，7-甲氧基-2-硝基萘并[2,1-b]呋喃（R7000）在大肠杆菌lacI基因中诱导的突变谱：SOS诱变的影响，《癌变》，1996年，第17卷，第2543 - 2550页]来看，22个（85%）发生在观察到终止产物的GC碱基对处。其他诱变事件涉及与形成复制阻断的G核苷酸相邻的AT碱基对。因此，所有诱变事件都发生在形成复制阻断的G核苷酸处或其附近。尽管不能排除某些诱变事件是由于未检测到的不阻断复制的DNA损伤所致，但这些结果强烈表明，阻断DNA复制的鸟苷 - DNA加合物是R7000产生的大部分诱变事件的原因。R7000在DNA序列中大多数鸟苷残基处形成加合物的强大能力，可能至少部分解释了其极强的遗传毒性特性。
