RecBCD enzyme switches lead motor subunits in response to chi recognition.
RecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, chi, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCD(K177Q) mutant with a defective RecD motor. RecBCD(K177Q) paused at chi but did not change its translocation velocity. RecBCD(K177Q) translocated at the same rate as the wild-type post-chi enzyme, implicating RecB as the lead motor after chi. P1 nuclease treatment eliminated the wild-type enzyme's velocity changes, revealing a chi-containing ssDNA loop preceding chi recognition and showing that RecD is the faster motor before chi. We conclude that before chi, RecD is the lead motor but after chi, the slower RecB motor leads, implying a switch in motors at chi. We suggest that degradation of foreign DNA needs fast translocation, whereas DNA repair uses slower translocation to coordinate RecA loading onto ssDNA.
Spies
Maria
M
Section of Microbiology, University of California, Davis, CA 95616-8665, USA.
Amitani
Ichiro
I
Baskin
Ronald J
RJ
Kowalczykowski
Stephen C
SC
D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009690:Nucleic Acid Conformation; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D020409:Molecular Motor Proteins; D021122:Protein Subunits; D043211:Exodeoxyribonuclease V; D015719:Single-Strand Specific DNA and RNA Endonucleases