Efficient coupling of ATP hydrolysis to translocation by RecQ helicase.
Helicases are ubiquitous enzymes that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) during essential processes such as replication, transcription, or repair. The Escherichia coli RecQ protein is a 3' to 5' helicase, which functions in the processes of homologous recombination and replication fork restart. Here, we analyzed the relationship between ATP hydrolysis by RecQ and its translocation on ssDNA. We monitored a single round of RecQ translocation on ssDNA by measuring the rates of inorganic phosphate release during translocation, and the dissociation of RecQ from ssDNA. We find that RecQ translocates with a rate of 16( ± 4) nucleotides/s and moves on average only 36( ± 2) nucleotides before dissociating. Fitting to an n-step kinetic model suggests that the helicase displays a nonuniform translocation mechanism in which it moves approximately five nucleotides rapidly before undergoing a rate-limiting kinetic slow step. Unexpectedly, RecQ requires a length of 34( ± 3) nucleotides to bind and translocate on ssDNA. This large site size suggests that several monomers are required to bind DNA prior to translocation. Energetically, the RecQ helicase couples the hydrolysis of one ATP molecule to the translocation of more than one nucleotide (1.6 ± 0.3). Thus, our data show that RecQ translocates on ssDNA by efficiently coupling the hydrolysis of one ATP molecule into structural alterations that result in movement of approximately two nucleotides, presumably by an inchworm mechanism. These attributes are consistent with the function of RecQ in recombination and replication.
Department of Microbiology, Department of Molecular and Cellular Biology, Graduate Group in Biophysics, University of California, Davis, CA 95616-8665, USA.
Behzad
B
Rad
Stephen C
SC
Kowalczykowski
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D053484:RecQ Helicases