title: Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase.
abstract: Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V(un)) by the velocity of translocation on ssDNA in nucleotides per second (V(trans)). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the "active" range. In the case of Dda, the average DNA unwinding velocity was 257±42 bp/s, and the average translocation velocity was 267±15 nt/s. The V(un)/V(trans) value of 0.96 places Dda in a unique category of being an essentially "perfectly" active helicase.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 516, Little Rock, AR 72205, USA.
authors.forename: Alicia K
authors.initials: AK
authors.lastname: Byrd
authors.forename: Dennis L
authors.initials: DL
authors.lastname: Matlock
authors.forename: Debjani
authors.initials: D
authors.lastname: Bagchi
authors.forename: Suja
authors.initials: S
authors.lastname: Aarattuthodiyil
authors.forename: David
authors.initials: D
authors.lastname: Harrison
authors.forename: Vincent
authors.initials: V
authors.lastname: Croquette
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Raney
mesh_terms: D001482:Base Composition; D004265:DNA Helicases / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D021381:Protein Transport; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004270:DNA, Circular; D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D014764:Viral Proteins; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4