Modulation of MutS ATP hydrolysis by DNA cofactors
Modulation of MutS ATP hydrolysis by DNA cofactors
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DOI：
10.1021/bi992286u
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发表时间：
2000-03-21
期刊：
影响因子：
2.9
通讯作者：
Modrich, P
中科院分区：
文献类型：
--
作者：
Bjornson, KP;Allen, DJ;Modrich, P
大肠杆菌MutS蛋白是错配修复所必需的，具有遵循Michaelis-Menten动力学的缓慢ATP酶活性。在37 ℃下，ATP水解的稳态周转率为1.0 +/- 0.3 min（-1）/单体当量，K-m为33 +/- 6 μ M。水解被ATP类似物AMPPNP和ATP γ S竞争性抑制，Ki，值为4 μ M，在这两种情况下，并通过ADP与a. K-i为40 μ M。ATP水解的速率被短的异源和同源双链DNA刺激2-5倍。对于含有错配碱基对的寡脱氧核苷酸双链体，产生半数最大刺激的DNA辅因子的浓度最低。前稳态化学淬灭分析表明，ADP形成的游离MutS，这是由78分钟（-1）的速率常数，表明水解后发生的稳态反应的限速步骤的亚化学计量的初始爆发。MutS与同源双链DNA的预结合不改变爆发动力学或振幅，但仅增加了爆发速率。与此相反，异源双链DNA的蛋白质的结合废除了ADP形成的爆发，表明限速步骤现在发生在水解之前。凝胶过滤分析表明MutS二聚体以浓度依赖性方式组装成更高级的寡聚体。的方式，ATP结合转移这种平衡，有利于组装。MutS寡聚体内亚基的不等价性这些结果以及动力学发现表明了ATP水解和DNA结合。
Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics, At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K-m of 33 +/- 6 mu M Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATP gamma S, with Ki, values of 4 mu M in both cases, and by ADP with a. K-i of 40 mu M. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest, for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the s t.eadystate rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent. manner, and that ATP binding shifts this equilibrium to favor assembly. nonequivalence of subunits within a MutS oligomer These results, together with kinetic findings, indicate with respect to ATP hydrolysis and DNA binding.