Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure.
Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.
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, 301 University Boulevard, Galveston, Texas 77555-1053, USA.
Rajendran
S
S
Bujalowski
W
W
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000378:metabolism; D053488:DnaB Helicases; D004926:Escherichia coli; D005454:Fluorescence Polarization; D046911:Macromolecular Substances; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate; D004265:DNA Helicases; D053488:DnaB Helicases