Strauss B S
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
Cancer Surv. 1985;4(3):493-516.
A system for the determination of the specificity of incorporation opposite lesions during DNA synthesis past damaged bases has been used as a model of mutation. The system, based on the dideoxynucleotide sequence method, uses lesions in the template strand as chain terminators. As a first approximation, such lesions constitute non-instructive sites in the DNA. DNA synthesis catalysed by bacteriophage T4 DNA polymerase terminates one nucleotide 3' to the lesion on the template strand. With other polymerases, synthesis may terminate opposite the lesion. The details of termination site depend on the enzyme, the metal ion (Mg2+ or Mn2+), the lesion and the particular nucleotide sequence. The sequence effect for termination on normal templates has two components, one ascribable to secondary structure, the other intrinsic to the sequence itself. DNA molecules terminated before lesions may be used as substrates in 'second stage' reactions in which elongation can be detected on the addition of particular dNTPs (deoxynucleoside triphosphates) to a reaction mixture. The specificity of elongation depends on the polymerase, on the activity of the 3'--greater than 5' editing nuclease and on the particular lesion. DNA polymerases have a preference for the addition of purines, particularly adenine, opposite non-instructional sites. This preference suggests an explanation for the specificity of base substitution mutations: treatments that produce non-instructional sites from purines will lead to transversions, treatments that affect pyrimidines lead to transitions. Even though a base is added opposite a lesion, further elongation may be rate limiting. Whether or not elongation occurs is dependent on the sequence 5' to the lesion on the template strand. The interactions of the factors affecting bypass: polymerase, lesion and sequence, may well result in an idiosyncratic behaviour for each mutable site.
一种用于确定DNA合成绕过受损碱基时在损伤位点对面掺入特异性的系统已被用作突变模型。该系统基于双脱氧核苷酸序列法，利用模板链中的损伤作为链终止剂。初步估计，此类损伤在DNA中构成非指令性位点。由噬菌体T4 DNA聚合酶催化的DNA合成在模板链上损伤位点的3'端一个核苷酸处终止。对于其他聚合酶，合成可能在损伤位点对面终止。终止位点的细节取决于酶、金属离子（Mg2+或Mn2+）、损伤以及特定的核苷酸序列。正常模板上终止的序列效应有两个组成部分，一个归因于二级结构，另一个是序列本身固有的。在损伤之前终止的DNA分子可在“第二阶段”反应中用作底物，在该反应中，向反应混合物中添加特定的脱氧核苷三磷酸（dNTP）时可检测到延伸。延伸的特异性取决于聚合酶、3'→5'编辑核酸酶的活性以及特定的损伤。DNA聚合酶倾向于在非指令性位点对面添加嘌呤，尤其是腺嘌呤。这种倾向为碱基替代突变的特异性提供了解释：从嘌呤产生非指令性位点的处理将导致颠换，影响嘧啶的处理导致转换。即使在损伤位点对面添加了一个碱基，进一步的延伸可能也是限速的。延伸是否发生取决于模板链上损伤位点5'端的序列。影响绕过的因素（聚合酶、损伤和序列）之间的相互作用很可能导致每个可变位点出现特异行为。
