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首页> 外文期刊>Chemistry: A European journal >Direct Monitoring Kinetic Studies of DNA Polymeraes Reactions on a DNA-Immobilized Quartz-Crystal Microbalance
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Direct Monitoring Kinetic Studies of DNA Polymeraes Reactions on a DNA-Immobilized Quartz-Crystal Microbalance

机译：在DNA固定的石英晶体微天平上直接监测DNA聚合反应的动力学研究

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摘要

Catalytic reactions of DNA Polymerase I from E.coli (Klenow frag-ment,KF)were monitored directly with a template/primer(40/25-or 75/25-mer)-immobilized 27-MHz quartz-crystal mi-crobalance(QCM).The 27-MHz QCMis a very sensitive mass-measuring de-vice in aqueous solution,as the frequen-cy decreases linearly with increasing mass on the QCM electrode at the nanogram level.Three steps in polymer-ase reactions which include 1) binding of DNA polymerase to the primer on the QCM(mass increase); 2) elongation of complementary nucleotides along the template(mass increase); and 3)release of the enzyme from the completely polymerized DNA (mass decrease),could be monitored continuously from the time dependencies of QCM frequen-cy changes.The binding constant (K_a) of KF to the template/primer DNA was 10~8M~(-1) (k_(on)=10~5M~(-1)s~(-1) and K_(off)=10~(-3)s~(-1)),and decreased to 10~6M~(-1) (K'_(on)=10~4M~(-1)s~(-1) and K'_(off)=10~(-2)s~(-1))) for completely polymerized DNA This is due to the 10-fold decrease in binding rate constant(K_(on)) and 10-fold increase in dissociation rate constant(K_(on)) for completed DNA strands K_a values de-pended slightly on the template and primer sequences.The kinetic parame-ters in the elongation process(K_(cat) and K_m) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.
机译：用固定有模板/引物（40 / 25-或75 / 25-mer）的27-MHz石英晶体微天平直接监测大肠杆菌（Klenow片段，KF）的DNA聚合酶I的催化反应。 27-MHz QCM是水溶液中非常敏感的质量测量装置，因为随着QCM电极质量的增加，其频率在纳克水平上呈线性下降。聚合物酶反应的三个步骤包括1 ）DNA聚合酶与QCM引物的结合（质量增加）; 2）互补核苷酸沿模板的延伸（质量增加）; 3）酶从完全聚合的DNA中释放（质量下降），可以根据QCM频率变化的时间依赖性连续监测。KF与模板/引物DNA的结合常数（K_a）为10〜8M 〜（-1）（k_（on）= 10〜5M〜（-1）s〜（-1）和K_（off）= 10〜（-3）s〜（-1）），并减小到10〜 6M〜（-1）（K'_（on）= 10〜4M〜（-1）s〜（-1）和K'_（off）= 10〜（-2）s〜（-1）））对于完全聚合的DNA，这是由于完整DNA链的结合速率常数（K_（on））降低10倍，解离速率常数（K_（on））增加10倍，K_a值略微降低延伸过程中的动力学参数（K_（cat）和K_m）仅略微依赖于DNA序列。当QCM频率变化时，KF催化的伸长过程中的修复过程也可以实时监控。

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