In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme
In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme
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DOI：
10.1006/jmbi.1998.2520
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发表时间：
1999-03-05
影响因子：
5.6
通讯作者：
Wallace, SS
中科院分区：
文献类型：
--
作者：
Hatahet, Z;Zhou, MX;Wallace, SS
使用体外选择系统解决了序列背景对DNA聚合酶绕过碱基丢失位点的能力的影响。在5'和3'侧均包含四个随机碱基的无碱基位点或四氢呋喃的寡核苷酸被用于Bs模板，用于通过3' -> 5'校正核酸外切酶活性熟练或缺陷的噬菌体T4 DNA聚合酶全酶合成。成功的旁路产物被纯化、亚克隆，并且对于所测试的四种聚合酶/损伤组合中的每一种确定大约100个亚克隆的序列。7 - 19%的旁路产物在随机化区域中含有1 - 3个核苷酸的缺失。不含缺失的Ln旁路产物，在整个随机化区域中容易注意到对某些核苷酸的偏好和对某些核苷酸的偏好。来自脱碱基位点的成功旁路产物的模板链在大多数随机化位置具有高频率的T，而来自四氢呋喃旁路产物的模板链在紧邻损伤的3'和5'侧的位置具有高频率的G。相同病变的成功旁路产品共享共有序列，但两个病变的旁路产品之间不共享。四氢呋喃有效旁路的共有序列在相对于病变的几个帧中过度呈现。T4 DNA聚合酶在有校对的情况下63%的时间插入A相对于无碱基位点，在没有校对的情况下79%的时间插入A相对于无碱基位点，其次是G > T > C，而在有校对和没有校对的情况下A相对于四氢呋喃的插入范围分别在93%和100%之间。最后，序列上下文影响的核苷酸插入相对的脱碱基位点的选择和共识序列，有利于纳入核苷酸以外的A被定义。(C)北京：科学出版社.
The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used Bs templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3' --> 5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. Ln bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G > T > C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined. (C) 1999 Academic Press.