Yang Ye, Dou Shuo-Xing, Ren Hua, Wang Peng-Ye, Zhang Xing-Dong, Qian Min, Pan Bing-Yi, Xi Xu Guang
Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China.
Nucleic Acids Res. 2008 Apr;36(6):1976-89. doi: 10.1093/nar/gkm1174. Epub 2008 Feb 14.
PcrA helicase, a member of the superfamily 1, is an essential enzyme in many bacteria. The first crystal structures of helicases were obtained with PcrA. Based on structural and biochemical studies, it was proposed and then generally believed that PcrA is a monomeric helicase that unwinds DNA by an inchworm mechanism. But a functional state of PcrA from unwinding kinetics studies has been lacking. In this work, we studied the kinetic mechanism of PcrA-catalysed DNA unwinding with fluorometric stopped-flow method under both single- and multiple-turnover conditions. It was found that the PcrA-catalysed DNA unwinding depended strongly on the PcrA concentration as well as on the 3'-ssDNA tail length of the substrate, indicating that an oligomerization was indispensable for efficient unwinding. Study of the effect of ATP concentration on the unwinding rate gave a Hill coefficient of approximately 2, suggesting strongly that PcrA functions as a dimer. It was further determined that PcrA unwound DNA with a step size of 4 bp and a rate of approximately 9 steps per second. Surprisingly, it was observed that PcrA unwound 12-bp duplex substrates much less efficiently than 16-bp ones, highlighting the importance of protein-DNA duplex interaction in the helicase activity. From the present studies, it is concluded that PcrA is a dimeric helicase with a low processivity in vitro. Implications of the experimental results for the DNA unwinding mechanism of PcrA are discussed.
PcrA解旋酶是超家族1的成员，是许多细菌中的一种必需酶。解旋酶的首批晶体结构就是通过PcrA获得的。基于结构和生化研究，有人提出并随后普遍认为PcrA是一种单体解旋酶，通过尺蠖机制解开DNA。但一直缺乏从解旋动力学研究中得出的PcrA的功能状态。在这项工作中，我们用荧光停流法在单周转和多周转条件下研究了PcrA催化的DNA解旋的动力学机制。发现PcrA催化的DNA解旋强烈依赖于PcrA的浓度以及底物的3'-单链DNA尾长，这表明寡聚化对于有效解旋是必不可少的。研究ATP浓度对解旋速率的影响得到的希尔系数约为2，强烈表明PcrA以二聚体形式发挥作用。进一步确定PcrA以4个碱基对的步长和解旋速率约为每秒9步解开DNA。令人惊讶的是，观察到PcrA解开12碱基对双链底物的效率远低于16碱基对的底物，这突出了蛋白质-DNA双链相互作用在解旋酶活性中的重要性。从目前的研究可以得出结论，PcrA在体外是一种低持续合成能力的二聚体解旋酶。讨论了实验结果对PcrA的DNA解旋机制的影响。
