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首页> 外文期刊>The FEBS journal >The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function
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The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function

机译：结核分枝杆菌UVRC的内在ATP酶活性对于其损伤的特异性DNA切口功能至关重要

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To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties ofMycobacterium tuberculosisUvrABC excinuclease complex. Furthermore, the biochemical properties ofEscherichia coliUvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instabilityin vitro. In addition, homology modelling ofM. tuberculosisUvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.
机译：为了确保基因组的稳定性，细菌进化出了一个DNA修复机制网络；其中，UvrABC依赖的核苷酸切除修复（NER）途径对于切割由外源性化学物质、紫外线辐射和细胞代谢副产物产生的各种大块加合物至关重要。然而，关于结核分枝杆菌UVRABC切核酸酶复合物的酶学性质知之甚少。此外，与UvrA和UvrB相比，大肠杆菌（EcUvrC）的生化特性尚不清楚，这可能是因为其在体外的可用性和/或活性不稳定性有限。此外，还对M进行了同源建模。结核性UVRC（MtUvrC）显示存在一个假定的ATP结合口袋，尽管其功能尚不清楚。为了阐明UvrC的生化特性，我们构建并纯化了野生型MtUvrC及其八个在ATP结合囊中携带突变的变体。来自DNA结合研究的数据表明，MtUvrC对含有气泡或荧光素dT部分的双链DNA表现出比荧光素加合单链DNA更高的亲和力。最值得注意的是，MtUvrC具有内在的UvrB非依赖性ATP酶活性，从而驱动受损DNA链的双重切割。相反，EcUvrC缺乏atp酶活性；然而，它仍能在与MtUvrC相当的水平上结合ATP。ATP酶缺陷变体映射到排列在MtUvrC ATP结合袋中的残基。对这些变体的进一步分析揭示了MtUvrC中ATP酶和DNA结合活性之间的功能分离。总之，这些发现揭示了细菌NER机制的功能多样性，以及UvrC中催化支架的进化范例。

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