Functional and structural heterogeneity of the DNA binding site of the Escherichia coli primary replicative helicase DnaB protein.
The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude approximately 10 nucleotides. ssDNA affinity for the strong subsite is approximately 3 orders of magnitude higher than that for the weak subsite. Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5'-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.
Jezewska
M J
MJ
Department of Human Biological Chemistry and Genetics and the Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1053, USA.
Rajendran
S
S
Bujalowski
W
W
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D053488:DnaB Helicases; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008836:Micrococcal Nuclease; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease; D004265:DNA Helicases; D053488:DnaB Helicases