Ren Hua, Dou Shuo-Xing, Rigolet Pascal, Yang Ye, Wang Peng-Ye, Amor-Gueret Mounira, Xi Xu Guang
School of Life Science, East China Normal University, Science Bld., 3663 North Zhongshan Rd., Shanghai 200062, PR China.
Nucleic Acids Res. 2007;35(18):6029-41. doi: 10.1093/nar/gkm544. Epub 2007 Aug 30.
RecQ family helicases are essential in maintaining chromosomal DNA stability and integrity. Despite extensive studies, the mechanisms of these enzymes are still poorly understood. Crystal structures of many helicases reveal a highly conserved arginine residue located near the gamma-phosphate of ATP. This residue is widely recognized as an arginine finger, and may sense ATP binding and hydrolysis, and transmit conformational changes. We investigated the existence and role of the arginine finger in the Bloom syndrome protein (BLM), a RecQ family helicase, in ATP hydrolysis and energy coupling. Our studies by combination of structural modelling, site-directed mutagenesis and biochemical and biophysical approaches, demonstrate that mutations of residues interacting with the gamma-phosphate of ATP or surrounding the ATP-binding sites result in severe impairment in the ATPase activity of BLM. These mutations also impair BLM's DNA-unwinding activities, but do not affect its ATP and DNA-binding abilities. These data allow us to identify R982 as the residue that functions as a BLM arginine finger. Our findings further indicate how the arginine finger is precisely positioned by the conserved motifs with respect to the gamma-phosphate.
RecQ家族解旋酶对于维持染色体DNA的稳定性和完整性至关重要。尽管进行了广泛的研究，但这些酶的作用机制仍知之甚少。许多解旋酶的晶体结构显示，在ATP的γ-磷酸附近有一个高度保守的精氨酸残基。这个残基被广泛认为是精氨酸指，可能感知ATP的结合和水解，并传递构象变化。我们研究了精氨酸指在RecQ家族解旋酶布鲁姆综合征蛋白（BLM）的ATP水解和能量偶联中的存在及作用。我们通过结构建模、定点诱变以及生化和生物物理方法相结合的研究表明，与ATP的γ-磷酸相互作用或围绕ATP结合位点的残基发生突变会导致BLM的ATP酶活性严重受损。这些突变也会损害BLM的DNA解旋活性，但不影响其ATP和DNA结合能力。这些数据使我们能够确定R982是作为BLM精氨酸指发挥作用的残基。我们的研究结果进一步表明了精氨酸指是如何通过保守基序相对于γ-磷酸精确定位的。
