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首页> 外文期刊>Biochemistry >Human DNA Polymerase a Uses a Combination of Positive and Negative Selectivity To Polymerize Purine dNTPs with High Fidelity
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Human DNA Polymerase a Uses a Combination of Positive and Negative Selectivity To Polymerize Purine dNTPs with High Fidelity

机译：人类DNA聚合酶a使用正负选择性的组合来以高保真度聚合嘌呤dNTPs

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DNA polymerases accurately replicate DNA by incorporating mostly correct dNTPs opposite any given template base.We have identified the chemical features of purine dNTPs that human pol alpha uses to discriminate between right and wrong dNTPs.Removing N-3 from guanine and adenine,two high-fidelity bases,significantly lowers fidelity.Analogously,adding the equivalent of N-3 to low-fidelity benzimidazole-derived bases(i.e.,bases that pol alpha rapidly incorporates opposite all four natural bases)and to generate 1-deazapurines significantly strengthens the ability of pol alpha to identify the resulting 1-deazapurines as wrong.Adding the equivalent of the purine N-l to benzimidazole or to 1-deazapurines significantly decreases the rate at which pol alpha polymerizes the resulting bases opposite A,C,and G while simultaneously enhancing polymerization opposite T.Conversely,adding the equivalent of adenine's C-6 exocyclic amine(N-6)to 1-and 3-deazapurines also enhances polymerization opposite T but does not significantly decrease polymerization opposite A,C,and G.Importantly,if the newly inserted bases lack N-l and N-6,pol alpha does not efficiently polymerize the next correct dNTP,whereas if it lacks N-3,one additional nucleotide is added and then chain termination ensues.These data indicate that pol alpha uses two orthogonal screens to maximize its fidelity.During dNTP polymerization,it uses a combination of negative(N-l and N-3)and positive(N-1 and N-6)selectivity to differentiate between right and wrong dNTPs,while the shape of the base pair is essentially irrelevant.Then,to determine whether to add further dNTPs onto the just added nucleotide,pol alpha appears to monitor the shape of the base pair at the primer 3'-terminus.The biological implications of these results are discussed.
机译：DNA聚合酶通过掺入与任何给定模板碱基相对的大多数正确的dNTP来准确复制DNA。我们已经鉴定了嘌呤dNTP的化学特征，人类pol alpha用来区分对错的dNTP。从鸟嘌呤和腺嘌呤中去除N-3，两个高类似地，在低保真度苯并咪唑衍生的碱基（即pol alpha快速结合了所有四个天然碱基的碱基）中添加相当于N-3的碱基，并生成1-deazapurines显着增强了保真度的能力。 pol alpha识别出生成的1-deazapurines是错误的。将等价的嘌呤N1加入苯并咪唑或1-deazapurines会显着降低pol alpha聚合与A，C和G相对的碱基的速率，同时会增强与A，C和G相对的碱基相反地​​，在1和3-脱氮嘌呤中加入当量腺嘌呤的C-6环胺（N-6）也会增强与T相反的聚合重要的是，如果新插入的碱基缺少Nl和N-6，pol alpha不会有效地聚合下一个正确的dNTP，而如果缺少N-3，则一个额外的核苷酸这些数据表明pol alpha使用两个正交筛选以使其保真度最大化。在dNTP聚合过程中，pol alpha使用了负数（Nl和N-3）和正数（N-1和N-6）的组合）区分正确和错误dNTP的选择性，而碱基对的形状基本上无关紧要。然后，为了确定是否在刚刚添加的核苷酸上添加其他dNTP，pol alpha似乎在引物上监控碱基对的形状3'-末端。讨论了这些结果的生物学含义。

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