DNA polymerase θ purified from human cells is a high-fidelity enzyme
DNA polymerase θ purified from human cells is a high-fidelity enzyme
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DOI：
10.1016/s0022-2836(02)00325-x
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发表时间：
2002-05-31
影响因子：
5.6
通讯作者：
Hübscher, U
中科院分区：
文献类型：
--
作者：
Maga, G;Shevelev, I;Hübscher, U
为了从人类细胞中鉴定非传统的DNA聚合酶，我们建立了一种特殊的方法来分离HeLa提取物，其基础是：(I)绕过DNA损伤，(Ii)对阿希迪林和针对Polaα的抑制性抗体的抗性，以及(Iii)对增殖细胞核抗原无反应。经过八个不同的层析步骤，获得了一种抗蚜虫啉的DNA聚合酶活性，它能够以同样的效率利用未受损或含有碱性位点的DNA。生物化学鉴定和免疫印迹分析证明其为人类DNA聚合酶theta(Hpoltheta)的同源物，其cDNA已通过与果蝇Mus308基因的同源性被克隆，但仍有待详细的生化特征鉴定。纯化的hpoltheta没有可检测到的解旋酶活性，具有3‘-gt；5’外切酶活性，表现出与迄今已知的任何其他真核DNA聚合酶明显不同的生化性质。误掺入和保真度分析表明：(I)hpoltheta能够有效地催化DNA合成通过一个基本位点；(Ii)hpoltheta表现出高保真度。我们的发现是根据hpoltheta(C)2002 Elsevier Science Ltd.提出的生理角色进行讨论的。保留所有权利。
With the aim to identify unconventional DNA polymerases from human cells, we have set up a special assay to fractionate HeLa extracts based on the ability (i) to bypass DNA lesions, (ii) to be resistant to aphidicolin and an inhibitory antibody against pol alpha and (iii) to be non-responsive to proliferating cell nuclear antigen. After eight different chromatographic steps, an aphidicolin-resistant DNA polymerase activity was obtained that was able to utilize either undamaged or abasic sites-containing DNA with the same efficiency. Biochemical characterization and immunoblot analysis allowed its identification as the human homologue of DNA polymerase theta (hpol theta), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol theta was devoid of detectable helicase activity, possessed a 3' --> 5' exonuclease activity and showed biochemical properties clearly distinct from any other eukaryotic DNA polymerase known so far. Misincorporation and fidelity assays showed that: (i) hpol theta was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol theta showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol theta (C) 2002 Elsevier Science Ltd. All rights reserved.