Does single-stranded DNA pass through the inner channel of the protein hexamer in the complex with the Escherichia coli DnaB Helicase? Fluorescence energy transfer studies.
The structure of the complex of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5' arm of the replication fork, without ATP hydrolysis, the distance between the 5' end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5' arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2-3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.
Jezewska
M J
MJ
Department of Human Biological Chemistry and Genetics and the Sealy Center for Structural Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1053, USA.
Rajendran
S
S
Bujalowska
D
D
Bujalowski
W
W
D001426:Bacterial Proteins; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004265:DNA Helicases; D053488:DnaB Helicases