Kinetics of oxidized cytosine repair by endonuclease III of Escherichia coli.
Kinetics of oxidized cytosine repair by endonuclease III of Escherichia coli.
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大肠杆菌内切核酸酶 III 氧化胞嘧啶修复的动力学。
DOI：
10.1021/bi970341y
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Essigmann,JM
中科院分区：
文献类型：
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作者：
Wang,D;Essigmann,JM
大肠杆菌的核酸内切酶III从DNA中切除广泛的氧化、水合和环片段化的嘧啶。比较了三种潜在致突变性氧化胞嘧啶损伤修复的动力学参数：5，6-二羟基-5，6-二氢-2 '-脱氧尿苷（尿嘧啶乙二醇或Ug）、5-羟基-2'-脱氧胞苷（5-ohC）和5-羟基-2 '-脱氧尿苷（5-ohU）。位点特异性修饰的40聚体寡核苷酸含有三个病变中的每一个在相同的序列背景下，化学合成或通过化学和酶的方法的组合。合成适当保护的5-ohC和5-ohU亚磷酰胺，并通过标准固相合成方法掺入寡核苷酸中。Ug的不稳定性使得有必要使用替代方法来制备含有Ug的类似40聚体。用OsO 4氧化含尿嘧啶的五聚体寡核苷酸，生成相应的含Ug的产物，然后将其连接到寡核苷酸支架上，生成40个碱基对的双链体。使用~（32）P标记的底物和基于凝胶电泳的测定，测定5-ohC、5-ohU和Ug切除的K和Vmax值。在本实验系统中，根据Vmax/Km的比值，修复效率的顺序为Ug > 5-ohC > 5-ohU。当病变对面的碱基配对从G变为A时，观察到适度的影响。
Endonuclease III ofEscherichia coliexcises a broad range of oxidized, hydrated and ring-fragmented pyrimidines from DNA. The kinetic parameters were compared for repair of three potentially mutagenic oxidized cytosine lesions: 5,6-dihydroxy-5,6-dihydro-2‘-deoxyuridine (uracil glycol or Ug), 5-hydroxy-2‘-deoxycytidine (5-ohC), and 5-hydroxy-2‘-deoxyuridine (5-ohU). Site-specifically modified 40-mer oligonucleotides containing each of the three lesions in the same sequence context were synthesized chemically or by a combination of chemical and enzymatic methods. Appropriately protected phosphoramidites of 5-ohC and 5-ohU were synthesized and incorporated into oligonucleotides by standard solid-phase synthetic methods. The lability of Ug made it necessary to use an alternative approach to prepare the analogous 40-mers containing Ug. An uracil containing pentamer oligonucleotide was oxidized with OsO4to generate the corresponding Ug containing product, which was then ligated into an oligonucleotide scaffold to generate 40 base pair duplexes. Using32P-labeled substrates and a gel electrophoresis based assay, the values ofKmandVmaxfor excision of 5-ohC, 5-ohU, and Ug were determined. In this experimental system, the order of repair efficiency is Ug > 5-ohC > 5-ohU based on ratios ofVmax/Km. Modest effects were observed when the base paired opposite the lesion was changed from G to A.