Peng W, Shaw B R
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708, USA.
Biochemistry. 1996 Aug 6;35(31):10172-81. doi: 10.1021/bi960001x.
The rate of UV-induced deamination of cytosine to uracil at a specific site in double-stranded (ds) DNA was monitored using a genetic reversion assay. M13mp2C141 ds DNA was exposed to 160 J/m2 UV (254 nm), incubated at 37 degrees C, pH 7.4, for various time intervals to allow for deamination, and treated with Escherichia coli photolyase in the presence of 365 nm light to reverse cyclobutane-type pyrimidine dimers. Upon transfection into uracil-glycosylase deficient (ung-) E. coli cells, the mutation (i.e., reversion) frequencies in the CCCC target sequence increased greatly with post-UV time of incubation at 37 degrees C, nearly doubling every day that the DNA had been held at 37 degrees C. After 8 days, the reversion frequencies had increased by two orders of magnitude upon transfection into ung- cells, relative to isogenic ung+ cells, indicating that most of the mutations arising in UV/photolyase-treated ds DNA were C-->T mutations mediated by a uracil intermediate. Sequencing of the revertants revealed that all mutations were single C-->T or tandem double CC-->TT mutations. An increasing percentage of tandem double CC-->TT mutations was found with longer post-UV incubation times, yet none occurred if the post-UV delay time step was omitted before photoreversal. After a 4-day delay between UV and photoreversal at 37 degrees C, greater than 84% of the total revertants had tandem double CC-->TT mutations. Thus, the generation of a tandem double mutation is a time-dependent process that arises in DNA after the initial UV exposure. The rate of appearance (with a pseudo-first-order rate constant ca. 10(-6) s-1) of tandem double mutations during incubation of UV-irradiated DNA is inconsistent with two random, independently occurring mutational events and suggests a concerted deamination of both residues in a tandem cytosine pyrimidine (C < > C) dimer. Considering that deamination in a C < > C dimer occurred here with a half-life of ca. 5 days, in contrast to the measured half-life of ca. 20,000 years for spontaneous (non-UV-treated) cytosine deamination for the same target, these studies show that the formation of pyrimidine dimers in DNA increases the rate of deamination by six orders of magnitude, leading to the accelerated formation of single C-->T and tandem double CC-->TT mutations.
使用遗传回复突变试验监测双链（ds）DNA中特定位点上紫外线诱导的胞嘧啶脱氨生成尿嘧啶的速率。将M13mp2C141双链DNA暴露于160 J/m²紫外线（254 nm）下，在37℃、pH 7.4条件下孵育不同时间间隔以进行脱氨反应，然后在365 nm光照下用大肠杆菌光解酶处理以逆转环丁烷型嘧啶二聚体。转染到尿嘧啶糖苷酶缺陷（ung-）的大肠杆菌细胞后，CCCC靶序列中的突变（即回复突变）频率随着在37℃下紫外线照射后的孵育时间大幅增加，DNA在37℃下保存的每一天频率几乎翻倍。8天后，相对于同基因的ung+细胞，转染到ung-细胞后的回复突变频率增加了两个数量级，这表明紫外线/光解酶处理后的双链DNA中出现的大多数突变是由尿嘧啶中间体介导的C→T突变。对回复突变体进行测序发现，所有突变均为单个C→T或串联双CC→TT突变。随着紫外线照射后的孵育时间延长，串联双CC→TT突变的比例不断增加，但如果在光逆转之前省略紫外线照射后的延迟时间步骤，则不会出现串联双CC→TT突变。在37℃下紫外线照射与光逆转之间延迟4天后，超过84%的总回复突变体具有串联双CC→TT突变。因此，串联双突变的产生是一个时间依赖性过程，在最初的紫外线照射后出现在DNA中。紫外线照射后的DNA在孵育过程中串联双突变的出现速率（假一级速率常数约为10⁻⁶ s⁻¹）与两个随机、独立发生的突变事件不一致，这表明串联胞嘧啶嘧啶（C<>C）二聚体中的两个残基发生了协同脱氨。考虑到这里C<>C二聚体中的脱氨半衰期约为5天，而相同靶标的自发（未紫外线处理）胞嘧啶脱氨半衰期约为20000年，这些研究表明DNA中嘧啶二聚体的形成使脱氨速率提高了六个数量级，导致单C→T和串联双CC→TT突变加速形成。
