title: Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.
abstract: DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding.
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Júlia
authors.initials: J
authors.lastname: Tóth
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Jack
authors.initials: J
authors.lastname: Bollins
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK mark.szczelkun@bristol.ac.uk.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D015263:Deoxyribonucleases, Type III Site-Specific / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D020349:Surface Plasmon Resonance
chemical_list: D000255:Adenosine Triphosphate; D004247:DNA; C093225:DNA restriction enzyme EcoPI; D015263:Deoxyribonucleases, Type III Site-Specific; D000251:Adenosine Triphosphatases