DEAMINATION OF CYTOSINE-CONTAINING PYRIMIDINE PHOTODIMERS IN UV-IRRADIATED DNA - SIGNIFICANCE FOR UV-LIGHT MUTAGENESIS
DEAMINATION OF CYTOSINE-CONTAINING PYRIMIDINE PHOTODIMERS IN UV-IRRADIATED DNA - SIGNIFICANCE FOR UV-LIGHT MUTAGENESIS
复制标题
DOI：
10.1074/jbc.270.41.24174
复制
发表时间：
1995-10-13
影响因子：
4.8
通讯作者：
LIVNEH, Z
中科院分区：
文献类型：
--
作者：
BARAK, Y;COHENFIX, O;LIVNEH, Z
环丁基嘧啶二聚体中的胞嘧啶快速脱氨基为尿嘧啶的认识提出了这种化学转化而不是酶促聚合酶错误是UV诱变中的主要诱变步骤的可能性。我们已经建立了一个灵敏的生物测定系统，使我们能够确定在质粒DNA中的环丁基嘧啶二聚体的胞嘧啶脱氨基的速率。这是通过体外紫外线照射和携带cro基因的质粒脱氨基，然后进行光复活，并通过尿嘧啶DNA N-糖基化酶缺陷的指示菌株中引起Cro（-）突变的能力来测定DNA中的尿嘧啶。DNA序列分析显示，29个Cro（-）突变体中有27个携带GC --> AT转换，正如从胞嘧啶脱氨基反应中预期的那样。紫外线照射质粒pOC 2中cro基因的胞嘧啶脱氨基作用在37 ℃下以一级动力学进行，速率为（3.9 +/- 0.6）x 10（-5）s（-1），对应于5 h的半衰期。生理盐条件下的半衰期增加到12小时，而降低pH值增加脱氨基。速率常数的温度依赖性产生的活化能为13.6 rt 3.3千卡/摩尔。这些动力学数据表明，脱氨基的胞嘧啶二聚体是太慢，发挥重要作用，在大肠杆菌的紫外诱变。然而，它可能在哺乳动物细胞中发挥重要作用，其中诱变过程较慢。
The realization that cytosine in cyclobutyl pyrimidine dimers rapidly deaminates to uracil raised the possibility that this chemical transformation, rather than an enzymatic polymerase error, is the major mutagenic step in UV mutagenesis. We have established a sensitive bioassay system that enabled us to determine the rate of deamination of cytosine in cyclobutyl pyrimidine dimers in plasmid DNA. This was done by in vitro UV irradiation and deamination of a plasmid carrying the cro gene, followed by photoreactivation, and assaying uracils in DNA by their ability to cause Cro(-) mutations in an indicator strain that was deficient in uracil DNA N-glycosylase, DNA sequence analysis revealed that 27 out of 29 Cro(-) mutants carried GC --> AT transitions, as expected from deamination of cytosine. Deamination of cytosines in the cro gene in UV-irradiated plasmid pOC2 proceeded at 37 degrees C with first-order kinetics, at a rate of (3.9 +/- 0.6) x 10(-5) s(-1), corresponding to a half-life of 5 h. Physiological salt conditions increased the half-life to 12 h, whereas decreasing the pH increased deamination. The temperature dependence of the rate constant yielded an activation energy of 13.6 rt 3.3 kcal/mol. These kinetics data suggest that deamination of cytosine-containing dimers is too slow to play an important role in UV mutagenesis in Escherichia coli. However, it is likely to play an important role in mammalian cells, where the mutagenic process is slower.