Yang Linjing, Beard William A, Wilson Samuel H, Broyde Suse, Schlick Tamar
Department of Chemistry and Courant, Institute of Mathematical Sciences, New York University and the Howard Hughes Medical Institute, 251 Mercer Street, New York, New York 10012, USA.
J Mol Biol. 2002 Apr 12;317(5):651-71. doi: 10.1006/jmbi.2002.5450.
The large-scale opening motion of mammalian DNA polymerase beta is followed at atomic resolution by dynamic simulations that link crystal "closed" and "open" conformations. The closing/opening conformational change is thought to be key to the ability of polymerases to choose a correct nucleotide (through "induced fit") and hence maintain DNA repair synthesis fidelity. Corroborating available structural and kinetic measurements, our studies bridge static microscopic crystal structures with macroscopic kinetic data by delineating a specific sequence, Phe272 ring flip, large thumb movement, Arg258 rotation with release of catalytic Mg2+, together with estimated time-scales, that suggest the Arg258 rearrangement as a limiting factor of large subdomain motions. If similarly slow in the closing motion, this conformational change might be restricted further when an incorrect nucleotide binds and thus play a role in pol beta's selectivity for the correct nucleotide. These results suggest new lines of experimentation in the study of polymerase mechanisms (e.g. enzyme mutants), which should provide further insights into mechanisms of error discrimination and DNA synthesis fidelity.
通过将晶体“闭合”和“开放”构象联系起来的动态模拟，以原子分辨率跟踪了哺乳动物DNA聚合酶β的大规模开放运动。闭合/开放构象变化被认为是聚合酶选择正确核苷酸（通过“诱导契合”）并因此维持DNA修复合成保真度能力的关键。我们的研究通过描绘特定序列、苯丙氨酸272环翻转、大拇指向运动、精氨酸258旋转并释放催化性镁离子，以及估计的时间尺度，证实了现有的结构和动力学测量结果，将静态微观晶体结构与宏观动力学数据联系起来，这表明精氨酸258重排是大亚结构域运动的限制因素。如果在闭合运动中同样缓慢，那么当错误的核苷酸结合时，这种构象变化可能会受到进一步限制，从而在β聚合酶对正确核苷酸的选择性中发挥作用。这些结果为聚合酶机制研究（如酶突变体）提出了新的实验方向，这应该能为错误识别和DNA合成保真度的机制提供进一步的见解。
