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首页> 外文期刊>Biochemistry >Determinants of DNA Mismatch Recognition within the Polymerase Domain of the Klenow Fragment
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Determinants of DNA Mismatch Recognition within the Polymerase Domain of the Klenow Fragment

机译：Klenow片段的聚合酶结构域内的DNA错配识别的决定因素。

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The Klenow fragment of Escherichia coli DNA polyerase I catalyzes template-directed synthesis of DNA and uses a separate 3'-5' exonuclease activity to edit misincorporated bases. The polymerase and exonuclease activities are contained in separate structural domains. In this study, nine Klenow fragment derivatives containing mutations within the polymerase domain were examined for their interaction with model primer-temperate duplexes. The partitioning of the DNA primer terminus between the polymerase and 3'-5' exonuclease active sites fo the mutant proteins was assessed by time-resolved fluorescence anisotropy, utilizing a dansyl fluorophore attached to the DNA. Mutation of N845 or R668 disrupted favorable interactions between the Klenow fragment and a duplex containing a matched terminal base pair but had litte effect when the terminus was mismatched. Thus, N845 and R668 are required for recognition of correct terminal base pairs in the DNA substrate. Mutation of N675, R835, R836, or R841 resulted in tighter polymerase site binding of DNA, suggesting that the side chains of these residues induce strain in the DNA and/or protein backbone a double mutant 9N675A/R841A) showed an even greater polymerase site partitioning than was displayed by either single mutation, indicating that such strain is additive. In both groups of mutant proteins, the ability of discriminate between duplexes containing matched or mismatched base pairs was impaired. In contrast, mutatio of K758 or Q849 had no effect on partitioning relative to wild type, regardless of DNA mismatch character. These results demonstrate that DNA mismathch recognitio is dependent on specific amino acid residues whthin the polymerase domain and is not governed solely by thermodynamic differences between correct and mismatched base pairs. Moreover, this study suggests a mechanism whereby the Klenow fragment is able to recognize polymerase errors following a misincorporatio event, leading to their eventual removal by the 3'-5' exonuclease activity.
机译：大肠杆菌DNA聚合酶I的Klenow片段催化DNA的模板指导合成，并使用单独的3'-5'核酸外切酶活性来编辑掺入错误的碱基。聚合酶和核酸外切酶活性包含在单独的结构域中。在这项研究中，检查了在聚合酶结构域内包含突变的九种Klenow片段衍生物与模型引物-温带双链体的相互作用。利用连接到DNA上的丹磺酰基荧光团，通过时间分辨的荧光各向异性来评估突变蛋白中DNA引物末端在聚合酶和3'-5'核酸外切酶活性位点之间的分配。 N845或R668的突变破坏了Klenow片段与包含匹配的末端碱基对的双链体之间的有利相互作用，但当末端错配时具有同窝效应。因此，需要N845和R668来识别DNA底物中的正确末端碱基对。 N675，R835，R836或R841的突变导致DNA的聚合酶位点结合更紧密，这表明这些残基的侧链会在DNA和/或蛋白质主链中诱导菌株，双突变体9N675A / R841A显示出更大的聚合酶位点突变比任一突变都显示，表明该菌株是可加的。在两组突变蛋白中，区分包含匹配或错配碱基对的双链体的能力均受损。相比之下，K758或Q849的突变对野生型的分配没有影响，而与DNA不匹配特征无关。这些结果表明，DNA错配识别依赖于聚合酶结构域中的特定氨基酸残基，而不仅仅由正确和错配碱基对之间的热力学差异决定。此外，这项研究提出了一种机制，通过该机制，Klenow片段能够在错误掺入事件之后识别聚合酶错误，从而最终通过3'-5'核酸外切酶活性将其清除。

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