title: Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.
abstract: We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
authors.forename: Paul C
authors.initials: PC
authors.lastname: Blainey
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Crampton
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
authors.forename: Tom
authors.initials: T
authors.lastname: Ellenberger
authors.forename: X Sunney
authors.initials: XS
authors.lastname: Xie
mesh_terms: D010582:Bacteriophage lambda / Q000201:enzymology*; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013816:Thermodynamics; D014764:Viral Proteins
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda