Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I.
Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I.
复制标题
体外跨损伤 DNA 复制经过大体积损伤的碱基序列依赖性，由 Pol I 的外切 Klenow 片段催化。
DOI：
10.1021/bi010005o
复制
发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Geacintov,NE
中科院分区：
文献类型：
--
作者：
Zhuang,P;Kolbanovskiy,A;Amin,S;Geacintov,NE
研究了碱基序列，特别是大DNA加合物侧翼不同的嘧啶类化合物对Escherichia colipol I(exo-)Klenow片段体外转译合成的影响。大块病变是由苯并[a]芘二醇环氧化物同分异构体[(+)-抗BPDE]与2-鸟嘌呤(G*)结合所致。在距5‘端19个碱基的位置用G*构建了四个不同的43碱基长寡核苷酸模板。除了位于G*两侧的嘧啶X或Y外，所有碱基都是相同的(序列上下文5‘-...XG*Y...，其中X，Y=C和/或T)。在所有情况下，加合物G*减慢引物延伸到G*之外的速度大于减慢与G*相对的dNTP插入的速度(A和G主要插入与G*相反的位置，其中A和G为A>；G)。根据X或Y的不同，完全病变搭桥的∼1.5−5(∼0.6−3.0%的搭桥效率)不同。5‘-侧的下游T侧翼G*而不是C有利于完全病变旁路，而上游C侧翼G*比T更有利。模板−中间产物错位导致的各种缺失产物尤其占优势(∼5.0−6.0%效率)，上游侧翼C，而3’-侧翼T降低缺失产物的水平(∼0.5−2.5%效率)。研究了(1)单dNTP插入与G*相对的单链dNTP插入和(2)单dNTP延伸到G*之外，或在所有四个dNTP存在的情况下，与G*相对的3‘端不同碱基(Z)的情况下的动力学。在具有上游C而不是T的序列的情况下，发现了异常有效的超出加合物的引物延伸效率(接近∼90%)，Z=T。这些效应可追溯到由于下游模板碱基序列对(从G*到4个−6碱基)与3‘端引物碱基及其5’侧翼碱基配对而引起的错位移码中间产物。后者取决于碱基Y和优先插入的与加合物相对的碱基。因此，下游模板序列以及G*两侧的碱基影响DNA跨损伤合成。
The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment ofEscherichia coliPol I (exo-) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] toN2-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5‘-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5‘-...XG*Y..., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G*, with A > G). Depending on X or Y, full lesion bypass differed by factors of ∼1.5−5 (∼0.6−3.0% bypass efficiencies). A downstream T flanking G* on the 5‘-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template−primer intermediates are particularly dominant (∼5.0−6.0% efficiencies) with an upstream flanking C, while a 3‘-flanking T lowers the levels of deletion products (∼0.5−2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3‘-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching ∼90%) was found with Z = T in the case of sequences with 3‘-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4−6 bases from G*) with the 3‘-terminal primer base and its 5‘-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.