Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase.
Helicases unwind dsDNA during replication, repair and recombination in an ATP-dependent reaction. The mechanism for helicase activity can be studied using oligonucleotide substrates to measure formation of single-stranded (ss) DNA from double-stranded (ds) DNA. This assay provides an 'all-or-nothing' readout because partially unwound intermediates are not detected. We have determined conditions under which an intermediate in the reaction cycle of Dda helicase can be detected by trapping a partially unwound substrate. The appearance of this intermediate supports a model in which each ssDNA product interacts with the helicase after unwinding has occurred. Kinetic analysis indicates that the intermediate appears during a slow step in the reaction cycle that is flanked by faster steps for unwinding. These observations demonstrate a complex mechanism containing nonuniform steps for a monomeric helicase. The potential biological significance of such a mechanism is discussed.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, Arkansas 72205, USA.
Robert L
RL
Eoff
Kevin D
KD
Raney
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D007700:Kinetics; D016018:Least-Squares Analysis; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; D004247:DNA; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4