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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• 作者：Colin G. Wu ; Fuqian Xie ; Timothy M. Lohman
• 关键词：ssDNA ; single©\stranded DNA ; dsDNA ; double©\stranded DNA ; chi ; crossover hotspot instigator ; PBP ; phosphate binding protein ; wt ; wild type
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 出版时间：26 October, 2012
• 年：2012
• 卷：423
• 期：3
• 页码：303-314
• 全文大小：1175 K

文摘

Escherichia coli RecBC, a rapid and processive DNA helicase with only a single ATPase motor (RecB), possesses two distinct single©\stranded DNA (ssDNA) 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 translocase (0.81 ¡À 0.05 ATP/nt), only its secondary translocase (1.12 ¡À 0.06 ATP/nt), or both translocases simultaneously (1.07 ¡À 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 ¡À 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.