Human apurinic/apyrimidinic endonuclease is processive
Human apurinic/apyrimidinic endonuclease is processive
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DOI：
10.1021/bi9907429
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发表时间：
1999-12-14
期刊：
影响因子：
2.9
通讯作者：
Strauss, PR
中科院分区：
文献类型：
--
作者：
Carey, DC;Strauss, PR
脱嘌呤/脱嘧啶核酸内切酶（AP endo）被认为在DNA氧化损伤的修复中起关键作用，并被认为启动碱基切除修复途径中大多数脱碱基位点的修复。AP内切在双链DNA中的脱碱基位点的5'处产生单个切口。在这项研究中，我们调查是否AP endo通过进行性或分配机制位于脱碱基位点。我们使用了线性多脱碱基位点底物（多联体），通过将相同的25个核苷酸单体单元（25聚体）连接在一起合成。我们首先确定，25-mer单体，从其中的多联体制备是由AP内切切口的方式类似于先前公布的49-mer基板与不同的序列。稳态参数K（m）和k（cat）和底物结合的单周转参数与先前发表的值相当。使用多脱碱基位点串联体，我们证明了AP endo能够在从其底物解离之前切割大约7至8个脱碱基位点，行进至少200个核苷酸。因此，AP内切酶与尿嘧啶DNA糖基化酶一样，以准进行性方式表现。合成能力可以从催化分离，因为合成能力在25 mM NaCl时最大，而裂解速率在125 mM盐时最大。简而言之，切刻活性最大化接近生理盐摩尔浓度，而持续合成能力是中等范围的生理盐浓度。后者可能受到离子强度微小变化的严格调节。
Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m)and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength.