Kinetic Mechanisms of the Nucleotide Cofactor Binding to the Strong and Weak Nucleotide-Binding Site of theEscherichia coliPriA Helicase. 2

Abstract

Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.

The Escherichia coli PriA Helicase Specifically Recognizes Gapped DNA Substrates

“…Interactions of the PriA Helicase with the Gapped DNA Substrates in the Presence of Nucleotide Cofactors-As mentioned above, the PriA helicase possesses two strong and weak nucleotide-binding sites, which dramatically affect the enzyme interactions with the ssDNA (25)(26)(27). In the next set of experiments, we address the effect of ADP and the ATP nonhydrolyzable analog, ATP␥S, on the recognition of the ssDNA gap by the PriA helicase.…”

“…5a and Table 1). On the other hand, the values of both K G Ϸ 2 ϫ The situation is very different for the considered gapped DNA substrate in the presence of the high ADP concentration, where both nucleotide-binding sites are saturated with the cofactor (25)(26)(27). Fluorescence titrations of the gapped DNA substrate, which has the ssDNA gap of 5 nucleotides, with the PriA helicase at two different nucleic acid concentrations, in buffer C (pH 7.0, 10°C), containing 3 ϫ 10 Ϫ3 M ADP, are shown in Fig.…”

“…5a. At this ADP concentration, the cofactor saturates only the strong nucleotide-binding site of the enzyme (25)(26)(27). The quantitative analysis of the titration curves has been analogously performed as described above for studies in the absence of the cofactors and shows that three PriA molecules associate with the gapped DNA.…”

“…However, unlike most of the helicases, the PriA protein has a significant affinity for the ssDNA in the absence of the nucleotide cofactor (20 -24). Moreover, what makes the PriA protein different from other well studied helicases is that the enzyme possesses two nucleotide-binding sites, a strong and a weak binding site, which profoundly differ in their affinities for the cofactors and their effects on the intrinsic affinity of the protein for the ssDNA (25)(26)(27). Furthermore, cooperative interactions between the two nucleotide-binding sites indicate an intricate communication between the sites (25).…”

“…This is surprising in light of the fact that the PriA helicase must specifically recognize the ssDNA gap formed at the damaged DNA site (16,17). Nothing is known about the role of two nucleotide-binding sites in the recognition process or the effect of the structure of the phosphate group of the nucleotides on the ssDNA gap recognition (25)(26)(27). Elucidation of the PriA-gapped DNA interactions and the role of nucleotide cofactors in the reaction are of fundamental importance for understanding of the enzyme mechanisms in both replication and recombination processes (6,(12)(13)(14)(15).…”