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首页> 外文期刊>Biochemistry >Structure of Purine-Purine Mispairs during Misincorporation and Extension by Escherichia coli DNA Polymerase I
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Structure of Purine-Purine Mispairs during Misincorporation and Extension by Escherichia coli DNA Polymerase I

机译：嘌呤-嘌呤错配在大肠杆菌DNA聚合酶I的掺入和延伸过程中的结构

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摘要

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated.The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA,the incoming dNTP,the template base,and both positions of the terminal base pair.A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base.During mispair formation,the k_(pol)/K_d values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP.These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation.During mispair extension,the only decrease in k_(pol)/K_d was associated with the G/G base pair in which 7-deazaguanine was in the template strand.These results as well as previous results [McCain et al.(2005) Biochemistry 44,5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension.These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation.During extension,however,the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.
机译：用高保真核酸外切酶活性失活的大肠杆菌DNA聚合酶I的高保真Klenow片段检查了嘌呤-嘌呤错配形成和延伸的机理。通过比较错配的动力学来检查嘌呤-嘌呤错配的结构。在DNA，进入的dNTP，模板碱基和末端碱基对的两个位置上的四个位置分别形成腺嘌呤和7-脱氮鸟嘌呤，鸟嘌呤和7-脱氮鸟嘌呤的形成。与7-脱氮嘌呤取代相关的速率降低表明在错配形成过程中，插入相对A的dATP（dATP / A）以及dATP / G和dGTP的k_（pol）/ K_d值dNTP中的去氮嘌呤使/ G降低大于10倍。这些结果表明，在错配对形成期间，新形成的碱基对呈Hoogsteen几何形状，而传入的dNTP在s中yn构象和反构象中的模板碱基。错配延伸期间，k_（pol）/ K_d的唯一减少与模板链中7-脱氮鸟嘌呤的G / G碱基对有关。以前的结果[McCain等人（2005）Biochemistry 44,5647-5659]，其中在G / A和G / G错误配对延伸过程中，在引物末端的鸟嘌呤3位与Arg668之间发现了氢键。结果表明，嘌呤-嘌呤错配是通过Hoogsteen几何构型形成的，其中dNTP处于顺式构象，而模板处于反式构象。然而，延伸时，引物末端的构象改变为反构型，而模板碱基可以为顺或反构象。

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