title: DNA contacts stimulate catalysis by a poxvirus topoisomerase.
abstract: Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5'-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.
authors: Salk Institute for Biological Studies, La Jolla, California 92037, USA.
authors: Y
authors: Y
authors: Hwang
authors: A
authors: A
authors: Burgin
authors: F
authors: F
authors: Bushman
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008977:Molluscum contagiosum virus / Q000201:enzymology*
chemical_list: D004279:DNA, Viral; D007288:Inosine; D004247:DNA; D004264:DNA Topoisomerases, Type I