Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase. Dda does not form stable oligomeric structures.
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.
Morris
P D
PD
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Tackett
A J
AJ
Babb
K
K
Nanduri
B
B
Chick
C
C
Scott
J
J
Raney
K D
KD
D000251:Adenosine Triphosphatases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014764:Viral Proteins
D004274:DNA, Recombinant; D014764:Viral Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4