Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.
Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.
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DOI：
10.1096/fj.201800899r
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发表时间：
2019-01
期刊：
影响因子：
--
通讯作者：
Kad NM
中科院分区：
文献类型：
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作者：
Barnett JT;Kad NM
核苷酸切除修复（NER）保护细胞免受各种类型的DNA损伤，主要是紫外线照射。在大肠杆菌中，损伤由两种关键酶识别：UvrA和UvrB。尽管进行了广泛的研究，UvrA的2 ATP酶结构域在NER中的作用仍然是难以捉摸的。结合单分子荧光显微镜和经典的生物化学方法，我们研究了核苷酸结合在UvrA的动力学循环中的作用。对UvrA的稳态ATP酶活性的测量表明，它在结合DNA时受到刺激（kcat 0.71-1.07/s）。尽管UvrA的能力，歧视损害，我们发现紫外线损伤的DNA不改变稳态ATP酶。为了了解损伤如何影响UvrA，我们在单分子水平上研究了它在各种核苷酸条件下与DNA的结合。我们已经发现，紫外线损伤和核苷酸辅因子影响的附着寿命的UvrA。在ATP和UV损伤的存在下，与未受损的DNA相比，寿命显著增加。为了调和这些观察结果，我们认为，UvrA使用负协同性之间的ATP酶位点，是门控损伤识别。只有在出现损坏的情况下，第二个部位才被激活，很可能是以顺序的方式。巴内特，J.T.，Kad，N. M.使用整体和单分子动力学了解DNA损伤检测和UvrA的ATP酶之间的耦合。
Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation, the role of UvrA’s 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA’s kinetic cycle. Measurement of UvrA’s steady-state ATPase activity shows it is stimulated upon binding DNA (kcat 0.71–1.07/s). Despite UvrA’s ability to discriminate damage, we find UV-damaged DNA does not alter the steady-state ATPase. To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the single molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA. To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential manner.—Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics.