STRAND SPECIFICITY IN THE INTERACTIONS OF ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASE DNAB PROTEIN WITH A REPLICATION FORK

The interactions of the Escherichia colt primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having e ither a single arm or both arms, have been studied using the thermodyn amically rigorous fluorescence titration techniques. This approach all ows 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 replicati on fork substrates, has also been characterized using the sedimentatio n velocity technique, To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replica tion fork substrates. In the presence of the ATP nonhydrolyzable analo g, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' a rm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm, ATP hydrolysis is not necessa ry for formation of the helicase-fork complex, The asymmetric interact ions 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 l ittle to the free energy of binding. The data indicate a rather passiv e role of the duplex parr of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the format ion of nonproductive complexes of the enzyme with the fork. Quantitati ve analysis of binding of the helicase to the two-arm fork substrate s hows that two DnaB hexamers can bind to the fork, with each single hex amer associated with a single arm of the fork. In this complex, the in trinsic affinity of the DnaB hexamer for the 5' arm ina two-arm fork i s not affected by the presence of the 3' arm, Moreover, the results sh ow that the 3' arm is in a conformation which makes it easily availabl e 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 fr om the 5' arm, The separation of both arms must be to such an extent t hat the 3' arm can bind an additional large DnaB hexamer. These result s reveal that the 3' arm is not engaged in thermodynamically stable in teractions with the helicase hexamer, when it is bound in its stationa ry complex to the 5' arm of the fork. The significance of the these re sults for a mechanistic model of the hexameric DnaB helicase action is discussed.