To determine if PriB affects the ATPase activity of PriA, we measured ATP hydrolysis catalyzed by 10 nM PriA in the Dabrafenib in vivo presence of 100 nM PriB (as monomers) and various concentrations of Fork 3 DNA (Figure 6A). This produces the same ratio of PriB to PriA that results in near maximal stimulation of PriA helicase activity (Figure 4A). Addition of

100 nM PriB (as monomers) yields a K m with respect to DNA of 3 ± 1 nM (Table 5). Thus, the presence of PriB has no significant effect on PriA’s K m with respect to DNA. We also examined the effect of PriB on PriA’s K m with respect to ATP (Figure 6B). With 10 nM PriA and in the absence of PriB, the K m with respect to ATP is 54 ± 19 μM (Table 5). Addition of 100 nM PriB (as monomers) yields a K m with respect to ATP of 70 ± 13 μM (Table 5). Thus, the presence of PriB has no significant effect on PriA’s K m with Z-VAD-FMK price respect to ATP. Table 5 Kinetic parameters for PriA’s ATPase activity in the presence and absence of PriB.   – PriB + PriB K m,DNA, nM 2 ± 1 3 ± 1 K m,ATP , μM 54 ± 19 70 ± 13 k cat , s -1 9 ± 1 14 ± 1 Kinetic parameters are mean values derived from at least three independent experiments and associated uncertainty values are one standard deviation of the mean. While PriB does not have a significant effect on PriA’s K m values for ATP or DNA, it does have a significant

effect on the value of k cat. In the presence of 100 nM PriB (as monomers), the k cat increases to 14 ± 1 s-1, indicating that PriB activates PriA’s ATPase activity (Figure 6 and Table 5). This observation lies in contrast to studies performed using E. coli PriA and PriB proteins that reveal no effect of PriB on the rate of PriA-catalyzed ATP hydrolysis [7]. Discussion In this study, we examined physical interactions within the DNA replication restart primosome of N. gonorrhoeae and the functional consequences of those interactions to gain insight into the biological significance of species variation in primosome protein function. Physical interactions within the DNA Nintedanib (BIBF 1120) replication restart primosome

of N. gonorrhoeae differ in several ways compared to those within the DNA replication restart primosome of E. coli. In E. coli, the PriA:PriB binary interaction is weak, while the PriB:DNA binary interaction is strong. In N. gonorrhoeae, these affinities have been reversed: the PriA:PriB binary interaction is strong, while the PriB:DNA binary interaction is weak. The crystal structure of N. gonorrhoeae PriB provides clues that could account for the low affinity PriB:DNA interaction. Analysis of the binding site for DNA reveals significantly reduced positive electrostatic surface charge potential relative to the analogous surface of E. coli PriB, and several aromatic residues of E. coli PriB that are known to play a role in binding ssDNA are not conserved in N. gonorrhoeae PriB [17, 18]. Furthermore, our results indicate that N. gonorrhoeae PriB shows little preference for binding specific DNA structures.