Stoichiometry and DNA unwinding by the bacteriophage T4 41:59 helicase.
The bacteriophage T4 41 protein is a replicative helicase that forms a hexamer in the presence of ATP and associates with the T4 59 protein. The stoichiometry of the 41:59 helicase complex and its mechanism for DNA unwinding have been investigated using steady-state and single-turnover kinetics. A partial duplex DNA fork containing two regions of single-stranded DNA (ssDNA) of 30 nucleotides each, and 30 base pairs served as the substrate. 59 was found to increase the steady-state unwinding rate of the substrate by 200-fold over the rate of 41 alone. Maximum unwinding occurred when 59 and 41 were equimolar, revealing a 1:1 stoichiometry for the complex. Varying 41 while holding 59 constant resulted in sigmoidal kinetics suggesting strong cooperativity for formation of the 41 hexamer and providing a lower limit for hexamer assembly of 65 nM. Substrates were prepared that contained a biotin-streptavidin block in either the leading or lagging strand of the duplex region of the substrate. The first order rate constant for unwinding was reduced only when the block was placed in the lagging strand of the DNA fork, indicating that the helicase interacts primarily with the lagging DNA strand.
Raney
K D
KD
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Carver
T E
TE
Benkovic
S J
SJ
D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; D001710:Biotin