Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork.
The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.
Jezewska
M J
MJ
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas 77555-1053, USA.
Rajendran
S
S
Bujalowski
W
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004265:DNA Helicases; D053488:DnaB Helicases