TY - JOUR
T1 - Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation
AU - Simon, Michael J.
AU - Sokoloski, Joshua E.
AU - Hao, Linxuan
AU - Weiland, Elizabeth
AU - Lohman, Timothy M.
N1 - Funding Information:
We thank Thang Ho for synthesis and purification of the DNA, Dr. Chris Ho for constructing the views of potential movements of the nuclease domain, Dr. Roberto Galletto for many fruitful discussions, suggestions, and comments on the ms, and Drs. Aaron Lucius and Gerald R. Smith for comments on the ms. This work was supported, in part, by NIH ( GM045948 ) to T.M.L. and by the American Cancer Society fellowship to J.S. ( PF-15-040-01-DMC ).
Publisher Copyright:
© 2016 Elsevier Ltd
PY - 2016/7/31
Y1 - 2016/7/31
N2 - Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80 bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB “arm” domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.
AB - Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80 bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB “arm” domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.
KW - SF1 helicase
KW - allostery
KW - fluorescence
KW - recombination
UR - http://www.scopus.com/inward/record.url?scp=84979536513&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2016.07.002
DO - 10.1016/j.jmb.2016.07.002
M3 - Article
C2 - 27422010
AN - SCOPUS:84979536513
SN - 0022-2836
VL - 428
SP - 2997
EP - 3012
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 15
ER -