The Primary and Secondary Translocase Activities within E. coli RecBC Helicase Are Tightly Coupled to ATP Hydrolysis by the RecB Motor

Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase

“…These mutations are in equivalent positions within helicase motif VI of the RecB and RecD motors (17,35). Previous studies showed that the DNA unwinding rate of RecB Y803H CD is slowed significantly (17), and this mutation also slows the rates of both the primary and secondary translocases within RecB Y803H C (20). As anticipated, the translocation rate of RecB Y803H CD in the 3Ј to 5Ј direction (Fig.…”

“…The primary translocase enables RecBC to move along ssDNA in the expected 3Ј to 5Ј direction, consistent with the directionality of RecB on ssDNA, whereas a secondary translocase facilitates translocation along the other DNA strand in the 5Ј to 3Ј direction, although this secondary translocase is not sensitive to the polarity of the ssDNA backbone (19). As such, RecBC can move along two non-complementary strands of ssDNA at the same rates in a concerted mechanism in which both translocases are tightly coupled to ATP hydrolysis within the RecB motor (20). Using an assay that enables direct measurement of the rates of ssDNA translocation of the RecBCD holoenzyme along each DNA strand in both the 3Ј to 5Ј and 5Ј to 3Ј directions, we show that the translocation rates along each DNA strand driven by the RecB and RecD motors are functionally coupled due to the action of the secondary RecBC translocase that operates within RecBCD.…”

“…Using a fluorescence assay that allows direct monitoring of translocation along each of the single strands, it has recently been shown that RecBC alone (without RecD) possesses two distinct translocase activities that are controlled by the single ATPase motor within RecB (19,20). The primary translocase enables RecBC to move along ssDNA in the expected 3Ј to 5Ј direction, consistent with the directionality of RecB on ssDNA, whereas a secondary translocase facilitates translocation along the other DNA strand in the 5Ј to 3Ј direction, although this secondary translocase is not sensitive to the polarity of the ssDNA backbone (19).…”

All motors have to decide is what to do with the DNA that is given them

“…Several of the most commonly used methods of monitoring the translocation of a motor protein are: a) measuring the arrival of the protein at a particular point along the DNA (19)(20)(21)(22)(23)(24); b) detecting the displacement of obstacles from DNA by the translocating motor (25)(26)(27); c) DNA-dependent ATPase activity of the motor (18,22,(27)(28)(29)(30)(31); d) translocation-induced changes in the structure/ topology of the DNA (32,33). While each approach has advantages and disadvantages, addressing the key questions typically involves a combined approach of several of these techniques.…”

“…Despite these differences, ATPase is tightly coupled to translocation for both translocase activities of the RecB motor (29). The coupling efficiency is (0.81 ± 0.05) ATP/nt when the 3′-5′ primary translocase is active, (1.12 ± 0.06) ATP/ nt when the secondary 5′-3′ translocase is active, and (1.07 ± 0.09) ATP/nt when both translocase activities are simultaneously active (29).…”