The primary and secondary translocase activities within E. coli RecBC helicase are tightly coupled to ATP hydrolysis by the RecB motor.
The primary and secondary translocase activities within E. coli RecBC helicase are tightly coupled to ATP hydrolysis by the RecB motor.
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DOI：
10.1016/j.jmb.2012.07.009
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发表时间：
2012-10-26
影响因子：
5.6
通讯作者：
Lohman, Timothy M.
中科院分区：
文献类型：
--
作者：
Wu, Colin G.;Xie, Fuqian;Lohman, Timothy M.
大肠杆菌 RecBC 是一种快速且持续的 DNA 解旋酶，仅具有单个 ATP 酶马达 (RecB)，具有两种不同的单链 (ss)DNA 转位酶活性，可在解开的双链 DNA 的每条链上进行操作。使用瞬态动力学测定法检测磷酸盐释放，我们表明，当仅使用其初级（0.81±0.05 ATP/nt）、仅使用其次级（1.12±0.06 ATP/nt）或同时使用两种易位酶（1.07±0.09 ATP/nt）沿着ssDNA易位时，RecBC水解相同量的ATP。 RecB (Y803H) 内的突变会减慢 RecBC 的初级易位速率，也会同等程度地减慢次级易位速率。这些结果表明，单个 RecB 马达的 ATPase 活性在紧密耦合的反应中驱动初级和次级 RecBC 易位酶。我们进一步表明，RecBC 在不断解旋双链 DNA 的同时也水解相同量的 ATP（0.95 ± 0.08 ATP/bp），这表明在 DNA 解旋过程中 RecBC 水解的大部分（可能是全部）ATP 用于促进 ssDNA 易位，而不是促进碱基对熔解。基于这些观察结果提出了 DNA 解旋模型。
E. coli RecBC, a rapid and processive DNA helicase with only a single ATPase motor (RecB), possesses two distinct single stranded (ss)DNA translocase activities that can operate on each strand of an unwound duplex DNA. Using a transient kinetic assay to detect phosphate release, we show that RecBC hydrolyzes the same amount of ATP when translocating along ssDNA using only its primary (0.81 ± 0.05 ATP/nt), only its secondary (1.12 ± 0.06 ATP/nt), or both translocases simultaneously (1.07 ± 0.09 ATP/nt). A mutation within RecB (Y803H) that slows the primary translocation rate of RecBC also slows the secondary translocation rate to the same extent. These results indicate that the ATPase activity of the single RecB motor drives both the primary and secondary RecBC translocases in a tightly coupled reaction. We further show that RecBC also hydrolyzes the same amount of ATP (0.95 ± 0.08 ATP/bp) while processively unwinding duplex DNA suggesting that the large majority, possibly all, of the ATP hydrolyzed by RecBC during DNA unwinding is used to fuel ssDNA translocation rather than to facilitate base pair melting. A model for DNA unwinding is proposed based on these observations.
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