A SENSITIVE GENETIC ASSAY FOR THE DETECTION OF CYTOSINE DEAMINATION - DETERMINATION OF RATE CONSTANTS AND THE ACTIVATION-ENERGY
A SENSITIVE GENETIC ASSAY FOR THE DETECTION OF CYTOSINE DEAMINATION - DETERMINATION OF RATE CONSTANTS AND THE ACTIVATION-ENERGY
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DOI：
10.1021/bi00462a015
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发表时间：
1990-03-13
期刊：
影响因子：
2.9
通讯作者：
SHAW, BR
中科院分区：
文献类型：
--
作者：
FREDERICO, LA;KUNKEL, TA;SHAW, BR
以前不可能确定37度时DNA中胞嘧啶的脱氨率。C，因为反应发生得很慢。我们在这里描述了一个敏感的遗传分析，以测量胞嘧啶脱胺率在DNA在单个胞嘧啶残基。该试验是基于对lacz . α基因突变体的还原。噬菌体M13mp2的基因编码序列，并采用缺乏尿嘧啶糖基酶的细菌菌株。该分析具有足够的灵敏度，使我们能够在给定地点检测到背景频率低至20万分之一的单一脱胺事件。通过这个实验，我们确定了单链DNA在温度范围从30到90度的胞嘧啶脱氨速率常数。C，然后计算单链DNA胞嘧啶脱氨的活化能为28 +-。1千卡每摩尔。为80.度。C，六个位点的脱氨速率常数变化小于3倍。为37.度。C， pH为7.4时单链和双链DNA胞嘧啶脱氨速率常数为1.1倍。10-10倍，约7倍。分别为每秒10-13次。(换句话说，单链DNA中胞嘧啶在37度时的半衰期。C大约是200年，而双链DNA大约是30000年)。因此，胞嘧啶被脱去。当出现在双螺旋结构中时，速度要慢140倍。这些和其他数据表明，脱氨速率强烈依赖于DNA结构和胞嘧啶质子化的程度。数据表明，扰乱DNA结构或促进胞嘧啶直接质子化的药物可能以生物学显著的速率诱导脱羧。该试验提供了一种直接检验假设的方法。
Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37.degree. C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ.alpha. gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90.degree. C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 .+-. 1 kcal/mol. At 80.degree. C, deamination rate constants at six sites varied by less than a factor of 3. At 37.degree. C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 .times. 10-10 and about 7 .times. 10-13 per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37.degree. C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years). Thus, cytosine is deaminated .apprx. 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.