Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.

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.

核苷酸切除修复（NER）保护细胞免受多种类型的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的两个ATP酶结构域在NER中的作用仍然难以捉摸。结合单分子荧光显微镜和经典的生化方法，我们研究了核苷酸结合在UvrA动力学循环中的作用。对UvrA稳态ATP酶活性的测量表明，它在结合DNA时受到刺激（kcat为0.71 - 1.07/秒）。尽管UvrA有辨别损伤的能力，但我们发现紫外线损伤的DNA并不会改变稳态ATP酶活性。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。我们发现紫外线损伤和核苷酸辅因子都会影响UvrA的结合持续时间。在有ATP和紫外线损伤的情况下，与未损伤的DNA相比，持续时间明显更长。为了协调这些观察结果，我们认为UvrA利用其ATP酶位点之间的负协同效应，这种协同效应由损伤识别控制。只有在存在损伤时，第二个位点才会被激活，很可能是以顺序的方式。——巴尼特，J.T.，卡德，N.M.利用整体和单分子动力学理解DNA损伤检测与UvrA的ATP酶之间的耦合