Miscoding properties of 3,N4-etheno-2'-deoxycytidine in reactions catalyzed by mammalian DNA polymerases.
Miscoding properties of 3,N4-etheno-2'-deoxycytidine in reactions catalyzed by mammalian DNA polymerases.
复制标题
哺乳动物 DNA 聚合酶催化反应中 3,N4-etheno-2-deoxycytidine 的错误编码特性。
DOI：
10.1021/bi961446o
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Grollman,AP
中科院分区：
文献类型：
--
作者：
Shibutani,S;Suzuki,N;Matsumoto,Y;Grollman,AP
我们研究了环外DNA加合物3，N4-亚乙基-2 '-脱氧胞苷（εdC）的错误编码特性，使用设计的实验系统检测和定量在DNA聚合酶α、β和δ催化的反应中由引物延伸产生的碱基取代和缺失。用εdC位点特异性修饰的寡核苷酸作为本研究的DNA模板。Pol α催化与εdC相反的dTMP和dAMP的掺入，伴随少量的dCMP和dGMP以及一些双碱基缺失。Pol β促进dCMP和dAMP的掺入，沿着少量的单碱基和双碱基缺失。Pol δ催化dTMP和较少量的dAMP和dGMP的掺入。通过稳态动力学分析确定了pol α和pol β催化反应中相对于εdC的核苷酸插入频率和从3 '引物末端延伸的链的频率。本研究的结果与引物延伸实验中获得的结果一致。体外测定的εdC的错误编码特性与哺乳动物细胞中位点特异性诱变实验中εdC→A颠换和εdC→T转换的观察结果一致（Moriyaet al.等人（1994）Proc. Acad. Sci. U.S.A.91，11899）。我们的结论是，从这项研究中，DNA聚合酶可能会有显着不同的错误编码的潜力和体外分析可以用来预测哺乳动物细胞中的环外DNA加合物的致突变特异性。
We have investigated the miscoding properties of the exocyclic DNA adduct, 3,N4-etheno-2‘-deoxycytidine (εdC), using an experimental system designed to detect and quantify base substitutions and deletions generated by primer extension in reactions catalyzed by DNA polymerases α, β, and δ. Oligodeoxynucleotides modified site-specifically with εdC were used as DNA templates for this study. Pol α catalyzed incorporation of dTMP and dAMP opposite εdC, accompanied by lesser amounts of dCMP and dGMP and some two-base deletions. Pol β promoted incorporation of dCMP and dAMP, along with small amounts of one-base and two-base deletions. Pol δ catalyzed incorporation of dTMP and lesser amounts of dAMP and dGMP. The frequency of nucleotide insertion opposite εdC and of chain extension from the 3‘-primer terminus in reactions catalyzed by pol α and pol β was established by steady-state kinetic analysis. Results of this study were consistent with those obtained in primer extension experiments. The miscoding properties of εdC determinedin vitroare consistent with observations of εdC→A transversions and εdC→T transitions in site-specific mutagenesis experiments in mammalian cells (Moriyaet al.(1994)Proc. Natl. Acad. Sci. U.S.A.91, 11899). We conclude from this study that DNA polymerases may differ significantly in their miscoding potential and thatin vitroanalysis can be used to predict mutagenic specificity of exocyclic DNA adducts in mammalian cells.