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J. Biol. Chem., Vol. 276, Issue 23, 19691-19698, June 8, 2001
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From the Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences,
Little Rock, Arkansas 72205
The active form of many helicases is oligomeric,
possibly because oligomerization provides multiple DNA binding sites
needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the
formation of an oligomeric species. The specific activity for ssDNA
stimulated ATPase activity was independent of Dda concentration. Dda
was mutated to produce an ATPase-deficient protein (K38A Dda) by
altering a residue within a conserved, nucleotide binding loop. The
helicase activity of K38A Dda was inactivated, although DNA binding
properties were similar to Dda. In the presence of limiting DNA
substrate, the rate of unwinding by Dda was not changed; however, the
amplitude of product formation was reduced in the presence of
increasing concentrations of K38A Dda. The reduction was between that
expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no
reduction in the observed rate for strand separation. Taken together,
these results indicate that oligomerization of Dda is not required for
DNA unwinding.
To whom correspondence and reprint requests should be
addressed. Tel.: 501-686-5244; Fax: 501-686-8169;
E-mail: raneykevind@exchange.uams.edu.
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