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首页> 外文期刊>Biochemistry >Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I.
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Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I.

机译：DNA聚合酶I通过无碱基位点类似物旁路合成的机理。

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摘要

Bypass synthesis by DNA polymerase I was studied using synthetic 40-nucleotide-long gapped duplex DNAs each containing a site-specific abasic site analog, as a model system for mutagenesis associated with DNA lesions. Bypass synthesis proceeded in two general stages: a fast polymerization stage that terminated opposite the abasic site analog, followed by a slow bypass stage and polymerization down to the end of the template. The position of the 3'-terminus of the primer relative to the absic site analog did not affect bypass synthesis in the range of -1 to -5. In contrast, bypass synthesis increased with the distance of the 5'-boundary of the gap from the lesion for up to 3-fold in the range of +1 to +9. Bypass synthesis was severely inhibited by moderate concentrations of salts, and under conditions that were optimal for the synthetic activity of DNA polymerase I (100 mM K+), bypass synthesis was completely inhibited (< 0.02% bypass). Elimination of the 3'-->5' proofreading exonuclease activity of thepolymerase, by using a mutant DNA polymerase, caused a dramatic 10-60-fold increase in bypass synthesis. Determination of the kinetic parameters for insertion opposite the abasic site analog revealed a strong preference for the insertion of dAMP, dictated by a lower Km and a higher kcat as compared to the other nucleotides. The rate of bypass was increased by omitting one or two dNTPs, most likely due to the facilitation of the polymerization past the lesion.
机译：使用合成的40个核苷酸长的带缺口的双链DNA（每个都包含一个位点特异性无碱基位点类似物）作为与DNA损伤相关的诱变的模型系统，研究了DNA聚合酶I的旁路合成。旁路合成过程分为两个大致阶段：快速聚合阶段（与无碱基位点类似物相对）终止，然后是缓慢的旁路阶段，直至模板末端聚合。相对于绝对位点类似物，引物的3'-末端的位置在-1至-5的范围内不影响旁路合成。相比之下，旁路合成随着间隙的5'边界距病变而增加，在+1至+9范围内可达3倍。中等浓度的盐会严重抑制旁路合成，在最适合DNA聚合酶I（100 mM K +）合成活性的条件下，旁路合成会被完全抑制（<0.02％旁路）。通过使用突变型DNA聚合酶消除聚合酶的3'-> 5'校对核酸外切酶活性，导致旁路合成过程中戏剧性地增加了10-60倍。确定与无碱基位点类似物相对的插入动力学参数表明，与其他核苷酸相比，较低的Km和较高的kcat决定了对dAMP插入的强烈偏好。通过省略一或两个dNTPs可以增加旁路的速度，这很可能是由于病变后的聚合反应促进了。

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