ATPase Cycle and DNA Unwinding Kinetics of RecG Helicase

The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.

超家族2细菌解旋酶RecG是一种单体酶，通过逆转停滞复制叉参与DNA修复过程。该解旋酶需以特异性且高效的方式发挥作用，以防止复制叉发生崩解。我们的研究证实，RecG可紧密且快速地结合模拟停滞复制叉的四链寡核苷酸连接结构。该解旋酶每水解一分子ATP，即可解旋约4个碱基长度的此类DNA连接结构。为深入解析其作用机制，我们采用荧光停流与猝灭流技术，检测了RecG结合DNA连接结构时其ATP酶循环中的单个反应步骤。全程使用荧光ATP类似物mantATP，以确定循环中的限速步骤、DNA对反应的影响以及循环内的主要状态。在可行条件下，我们同时使用未标记的ATP开展实验，以验证该作用机制。实验数据表明，ATP水解的化学反应步骤是整个循环的限速步骤，且结合的DNA可显著加速该步骤。ADP释放速率与ATP裂解速率相近，因此结合态的ATP与ADP将成为ATP循环中的主要存在状态。本研究还提供了相关证据，表明介导DNA解旋的主要结构重排过程与上述循环状态紧密关联。