Accelerated deamination of cytosine residues in UV-induced cyclobutane pyrimidine dimers leads to CC->TT transitions
Accelerated deamination of cytosine residues in UV-induced cyclobutane pyrimidine dimers leads to CC->TT transitions
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DOI：
10.1021/bi960001x
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发表时间：
1996-08-06
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：
Peng, W;Shaw, BR
使用遗传回复测定法监测双链 (ds) DNA 中特定位点处 UV 诱导的胞嘧啶脱氨基为尿嘧啶的速率，将 M13mp2Cl41 ds DNA 暴露于 160 J/m(2) UV (254 nm)，在 37 摄氏度、pH 7.4 下孵育不同的时间间隔以允许脱氨基，并在存在365 nm 光可逆转环丁烷型嘧啶二聚体。转染到尿嘧啶糖基酶缺陷型 (ung(-)) 大肠杆菌细胞后，CCCC 靶序列中的突变（即回复）频率在 37 摄氏度下进行 UV 培养后大大增加，几乎每天都在 37 摄氏度下加倍。8 天后，相对于同基因的，转染到 ung(-) 细胞后，回复频率增加了两个数量级ung(+)细胞，表明UV/光裂解酶处理的双链DNA中产生的大多数突变是由尿嘧啶中间体介导的C-->T突变。回复体的测序显示，所有突变都是单 C-->T 或串联双 CC-->TT 突变，随着 UV 后孵育时间的延长，发现串联双 CC-TT 突变的百分比增加。然而，如果在光反转之前省略紫外线后延迟石灰步骤，则不会发生任何情况。在 37°C 的紫外线和光逆转之间延迟 4 天后，超过 84% 的总回复体具有串联双 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在 DNA 中出现。在紫外线照射的 DNA 孵育过程中，串联双突变的罕见出现（伪一级速率常数约为 10(-6) s(-1)）与两个随机、独立发生的突变事件不一致，表明串联胞嘧啶嘧啶（C >$($) 超过条 C）二聚体中两个残基的协同脱氨基作用。考虑到 C >$($) 超过 C 二聚体的脱氨作用发生在此处，半衰期约为 1。对于同一靶标，自发（未经紫外线处理）胞嘧啶脱氨基作用的半衰期为 5 天，而测量的半衰期为 20 000 年，这些研究表明，DNA 中嘧啶二聚体的形成使脱氨基速率增加了六个数量级，导致单 C-T 和串联双 CC-TT 突变的加速形成。
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, M13mp2Cl41 ds DNA was exposed to 160 J/m(2) UV (254 nm), incubated al 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 transfect-ion into uracil-glycosylase deficient (ung(-)) E. coli cells, the mutation (i.e,, reversion) frequencies in the CCCC target sequence Increased greatly with post-UV lime of incubation al 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 lime 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 rare 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 >$($) over bar C) dimer. Considering that deamination in a C >$($) over bar 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.