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首页> 外文期刊>Biochemistry >p-Thiophenylalanine-Induced DNA Cleavage and Religation Activity of a Modified Vaccinia Topoisomerase IB
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p-Thiophenylalanine-Induced DNA Cleavage and Religation Activity of a Modified Vaccinia Topoisomerase IB

机译：对硫苯丙氨酸诱导的DNA切割和修饰的牛痘拓扑异构酶IB的连接活性。

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摘要

Vaccinia DNA topoisomerase IB is the smallest of the type IB topoisomerases. Because of its small size (314 amino acids) and target site specificity (5'(C/T)CCTTp~↓ sites), it constitutes an excellent model for studying the interaction of type IB enzymes with duplex DNA. In this study, p-thiophenylalanine was incorporated into the enzyme active site (position 274) by in vitro translation in the presence of a chemically misacylated tRNA. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate (k_(d) = 1 × 10~(-4) s~(-1)) was 30 times lower than for the wildtype enzyme (k(d) = 3 × 10~(-3) s~(-1)). In contrast, thiophenylalanine-induced DNA religation was faster than that of the wild-type enzyme. We propose that the change in kinetics reflects the difference in bond energies between the O-P and S-P bonds being formed and broken in the reactions catalyzed by the wild-type and modified enzymes. We also studied the effect of adding Mg~(2+) and Me to the wild-type and modified topoisomerases I. Divalent metal ions such as Mg~(2+) and Mn~(2+) increased DNA relaxation activity of the wild-type and modified enzymes. However, the pattern of increases failed to support the possibility that metal ion-heteroatom interaction is required for catalysis.
机译：痘苗DNA拓扑异构酶IB是IB型拓扑异构酶中最小的。由于其体积小（314个氨基酸）和靶位点特异性（5'（C / T）CCTTp〜↓位点），它是研究IB型酶与双链DNA相互作用的极佳模型。在这项研究中，对-硫代苯丙氨酸通过在化学错误酰化的tRNA存在下的体外翻译而被整合到酶的活性位点（274位）中。该修饰导致亲核酪氨酸OH基团被SH取代，保留了DNA拓扑异构酶活性，并且没有改变DNA切割位点。然而，修饰的拓扑异构酶以比野生型酶慢约16倍的速率实现超螺旋质粒DNA的松弛。硫代苯丙氨酸诱导的DNA裂解率（k_（d）= 1×10〜（-4）s〜（-1））比野生型酶（k（d）= 3×10〜（-3）低30倍）s〜（-1））。相反，硫代苯丙氨酸诱导的DNA连接要比野生型酶快。我们建议动力学的变化反映了在野生型和修饰的酶催化的反应中形成和破坏的O-P和S-P键之间键能的差异。我们还研究了将Mg〜（2+）和Me添加到野生型和修饰的拓扑异构酶I中的作用。Mg〜（2+）和Mn〜（2+）等二价金属离子增加了野生DNA的松弛活性型和修饰的酶。然而，增加的模式不能支持催化需要金属离子-杂原子相互作用的可能性。

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