Wang Hong, DellaVecchia Matthew J, Skorvaga Milan, Croteau Deborah L, Erie Dorothy A, Van Houten Bennett
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2006 Jun 2;281(22):15227-37. doi: 10.1074/jbc.M601476200. Epub 2006 Apr 4.
UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.
UvrB是细菌核苷酸切除修复中一种核心的DNA损伤识别蛋白，它与DNA的亲和力较弱，其ATP酶活性由UvrA和受损DNA激活。目前对UvrB调节DNA结合和ATP水解的机制了解甚少。利用原子力显微镜和生化分析，我们发现嗜热栖热芽孢杆菌UvrB（UvrBDelta4）的结构域4截短会导致蛋白质功能发生多种变化。蛋白质二聚化减少，平衡解离常数增加约8倍，同时DNA结合增加。结构域4的缺失导致UvrB的DNA结合模式从二聚体转变为单体，并且在未受损和受损单链DNA上的表观解离常数分别降低22倍和14倍，亲和力增加。有和没有单链DNA时，UvrBDelta4的ATP酶活性分别增加14倍和9倍，并且UvrBDelta4支持Cho在气泡DNA底物上进行不依赖UvrA的损伤特异性切割。我们提出，除了其先前发现的在调节蛋白质-蛋白质相互作用中的作用外，结构域4还是一个调节UvrB的DNA结合和ATP酶活性的自抑制结构域。
