Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase |
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Authors: | Eoff Robert L Raney Kevin D |
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Affiliation: | Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, Arkansas 72205, USA. |
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Abstract: | 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. |
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