Kappen L S, Goldberg I H
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1999 Jan 5;38(1):235-42. doi: 10.1021/bi982185g.
Under anaerobic conditions neocarzinostatin chromophore, an enediyne antibiotic, forms a covalent drug-DNA adduct on the 5' carbon of deoxyribose at a specific single site in a 2-nucleotide bulge, rather than strand cleavage, by a mechanism involving general base-catalyzed intramolecular drug activation to a reactive radical species. We have taken advantage of the selectivity of this reaction to prepare a single-stranded oligonucleotide containing a single drug adduct at a T residue and to study its effect on the template properties of the oligonucleotide in replicative synthesis, as followed by 5'-32P-labeled primer extension by several DNA polymerases. With the Klenow fragment of Escherichia coli DNA polymerase I, synthesis stops at the base immediately 3' to the adduct. The same enzyme, but lacking 3' to 5' exonuclease activity, permits synthesis to proceed by one additional nucleotide. This effect is enhanced when Mn2+ is substituted for Mg2+. T4, herpes simplex virus, and cytomegalovirus DNA polymerases all act like Klenow polymerase. Sequenase (exo-minus T7 DNA polymerase) is qualitatively similar to exo-minus Klenow polymerase but is more efficient in inserting a nucleotide opposite the lesion. With the small-gap-filling human DNA polymerase beta, which lacks intrinsic exonucleolytic activity, primer extension proceeds to the nucleotide opposite the lesion. However, when a gap was created opposite the lesion, polymerase beta adds as many as two additional nucleotides 5' to the adduct site. The fidelity of base incorporation opposite the lesion was not impaired, in contrast with adducts on DNA bases.
在厌氧条件下，新制癌菌素发色团（一种烯二炔类抗生素）通过一种涉及一般碱催化的分子内药物活化形成反应性自由基物种的机制，在2个核苷酸凸起处的特定单一位点的脱氧核糖5'碳上形成共价药物 - DNA加合物，而不是链断裂。我们利用了该反应的选择性，制备了在T残基处含有单个药物加合物的单链寡核苷酸，并研究其对复制合成中寡核苷酸模板性质的影响，随后通过几种DNA聚合酶进行5'-32P标记的引物延伸。使用大肠杆菌DNA聚合酶I的Klenow片段，合成在加合物紧邻的3'端碱基处停止。同样的酶，但缺乏3'至5'外切核酸酶活性，允许合成再进行一个核苷酸。当用Mn2+替代Mg2+时，这种效应会增强。T4、单纯疱疹病毒和巨细胞病毒DNA聚合酶的作用都类似于Klenow聚合酶。测序酶（无外切酶活性的T7 DNA聚合酶）在性质上与无外切酶活性的Klenow聚合酶相似，但在损伤位点对面插入核苷酸方面更有效。对于缺乏内在外切核酸酶活性的小间隙填充人DNA聚合酶β，引物延伸会进行到损伤位点对面的核苷酸处。然而，当在损伤位点对面产生一个间隙时，聚合酶β会在加合物位点5'端添加多达两个额外的核苷酸。与DNA碱基上的加合物不同，损伤位点对面碱基掺入的保真度没有受损。
