TY - JOUR
T1 - The primary and secondary translocase activities within E. coli RecBC helicase are tightly coupled to ATP hydrolysis by the RecB motor
AU - Wu, Colin G.
AU - Xie, Fuqian
AU - Lohman, Timothy M.
N1 - Funding Information:
This research was supported in part by National Institutes of Health grant GM045948 . We thank Thang Ho for synthesis and purification of DNA; Dr. Elizabeth Weiland for making the overexpression plasmid for RecB Y803H ; Josh Brettmann for preliminary studies of RecB Y803H C; Drs. C. Fischer, A. Lucius, and E. Tomko for discussions of data analysis; and Drs. G. Smith and D. Julin for providing plasmids and cell lines.
PY - 2012/10/26
Y1 - 2012/10/26
N2 - 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 ± 0.05 ATP/nt), only its secondary translocase (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.
AB - 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 ± 0.05 ATP/nt), only its secondary translocase (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.
KW - allostery
KW - double-strand break repair
KW - recombination
KW - translocation kinetics
KW - unwinding mechanism
UR - http://www.scopus.com/inward/record.url?scp=84867082338&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2012.07.009
DO - 10.1016/j.jmb.2012.07.009
M3 - Article
C2 - 22820092
AN - SCOPUS:84867082338
SN - 0022-2836
VL - 423
SP - 303
EP - 314
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -