DNA damage recognition of mutated forms of UvrB proteins in nucleotide excision repair.
DNA damage recognition of mutated forms of UvrB proteins in nucleotide excision repair.
复制标题
核苷酸切除修复中 UvrB 蛋白突变形式的 DNA 损伤识别。
DOI：
10.1021/bi035992a
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Lloyd,RStephen
中科院分区：
文献类型：
--
作者：
Zou,Yue;Ma,Huaxian;Minko,IrinaG;Shell,StevenM;Yang,Zhengguan;Qu,Youxing;Xu,Ying;Geacintov,NicholasE;Lloyd,RStephen
DNA修复蛋白UvrB在大肠杆菌核苷酸切除修复的逐步和顺序损伤识别中起着不可或缺的作用。我们以前的研究表明，UvrB是负责的化学损伤识别后，由UvrA介导的链打开。由于存在UvrA，在研究UvrB与加合物的直接相互作用时遇到了困难。我们在此报告，Y 95 W的一个单一的点突变，其中酪氨酸被替换为色氨酸的结果在UvrB突变体，能够有效地结合到结构特异性的DNA加合物，即使在UvrA的情况下。该突变体在UvrABC切口中完全起作用。通过荧光光谱法测定，在生理温度下，突变体-DNA加合物相互作用的解离常数小于100 nM。相比之下，在β-发夹中的其他残基处用色氨酸或苯丙氨酸的类似取代不赋予UvrB这种结合能力。对E. coliUvrB显示残基Y 95的芳环和仅Y 95直接指向DNA结合裂缝。我们还研究了UvrB对“正常”大体积BPDE-DNA和蛋白质-交联DNA（DPC）加合物的识别，以及β-发夹的芳香残基在识别这些损伤中的作用。Y 92 W的突变导致正常加合物的UVrABC切割效率明显降低，而DPC加合物的切割显著增加。我们的研究结果表明，Y 92可能与这两种类型的加合物的功能不同，而Y 95残基在稳定UvrB与DNA损伤的相互作用中起着独特的作用，最有可能是通过疏水堆积。
The DNA repair protein UvrB plays an indispensable role in the stepwise and sequential damage recognition of nucleotide excision repair inEscherichia coli. Our previous studies suggested that UvrB is responsible for the chemical damage recognition only upon a strand opening mediated by UvrA. Difficulties were encountered in studying the direct interaction of UvrB with adducts due to the presence of UvrA. We report herein that a single point mutation of Y95W in which a tyrosine is replaced by a tryptophan results in an UvrB mutant that is capable of efficiently binding to structure-specific DNA adducts even in the absence of UvrA. This mutant is fully functional in the UvrABC incisions. The dissociation constant for the mutant−DNA adduct interaction was less than 100 nM at physiological temperatures as determined by fluorescence spectroscopy. In contrast, similar substitutions at other residues in the β-hairpin with tryptophan or phenylalanine do not confer UvrB such binding ability. Homology modeling of the structure ofE. coliUvrB shows that the aromatic ring of residue Y95 and only Y95 directly points into the DNA binding cleft. We have also examined UvrB recognition of both “normal” bulky BPDE−DNA and protein−cross-linked DNA (DPC) adducts and the roles of aromatic residues of the β-hairpin in the recognition of these lesions. A mutation of Y92W resulted in an obvious decrease in the efficiency of UvrABC incisions of normal adducts, while the incision of the DPC adduct is dramatically increased. Our results suggest that Y92 may function differently with these two types of adducts, while the Y95 residue plays an unique role in stabilizing the interaction of UvrB with DNA damage, most likely by a hydrophobic stacking.