B) Unwinding

of 1 nM Fork 3 by 2 nM PriA in the presence of wild type N. gonorrhoeae PriB (circles) or PriB:K34A (squares). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. When we examined PriA helicase activity on Fork 3 in the presence of PriB:K34A, we found that levels of DNA unwinding are similar to those seen when wild type PriB is used to stimulate PriA (Figure 5B). Based on the value of the apparent dissociation constant for the interaction of PriB:K34A with ssDNA, and assuming that it is a reliable PU-H71 indicator of the affinity of PriB:K34A for DNA in the context of a ternary PriA:PriB:DNA complex, we would not expect the PriB:K34A variant to be interacting with DNA to a significant degree under the conditions of this DNA unwinding assay. It is particularly noteworthy that in E. coli, a PriB variant with severely weakened ssDNA binding ARN-509 activity (the W47,K82A double mutant) fails to stimulate the DNA unwinding activity of its cognate PriA to a significant degree [7]. Therefore, unless formation of a PriA:PriB:DNA ternary complex significantly enhances the DNA binding activity of N. gonorrhoeae PriB, our results suggest that ssDNA binding by N. gonorrhoeae PriB does not play a major role in N. gonorrhoeae PriB stimulation of its cognate PriA helicase. PriB Rigosertib purchase activates PriA’s ATPase activity PriA helicase

is thought to couple the energy released from hydrolysis of ATP to the unwinding of duplex DNA. Thus, we wanted to determine if N. gonorrhoeae PriB stimulation of PriA helicase activity involves PriA’s ability to hydrolyze ATP. To examine PriA’s ATPase activity, we used a spectrophotometric assay that couples PriA-catalyzed ATP hydrolysis to oxidation of NADH. This assay allowed us to measure steady-state PriA-catalyzed

ATP hydrolysis rates in the presence and absence of PriB. As expected, PriA’s ATPase activity is negligible in the absence of DNA (Figure 6A). The DNA dependence of PriA’s ATPase activity has been observed in E. coli as well [30], and likely reflects a mechanistic coupling of ATP hydrolysis and duplex DNA unwinding. Figure 6 PriA’s ATPase activity is however stimulated by DNA and by PriB. A) DNA-dependent ATP hydrolysis catalyzed by 10 nM PriA in the presence (circles) or absence (squares) of 100 nM PriB (as monomers). The DNA substrate is Fork 3. Measurements are reported in triplicate and error bars represent one standard deviation of the mean. B) Effect of ATP concentration on rates of ATP hydrolysis catalyzed by 10 nM PriA in the presence of 100 nM Fork 3 and in the presence (circles) or absence (squares) of 100 nM PriB (as monomers). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. With 10 nM PriA and in the absence of PriB, near maximal rates of ATP hydrolysis are observed with 10 nM Fork 3 (Figure 6A).