Contrary to find more expectations, the DNA binding activity of PriB shows little preference for specific DNA structures (Figure 2). The apparent dissociation constants range from 628 ± 95 nM (3′ Overhang) to 690 ± 51 nM (Fork 2), and the observed differences among apparent dissociation constants for the various DNA structures are insignificant given the experimental uncertainty of the

measurements (Table 2). This observation, together with the low affinity with which N. gonorrhoeae PriB binds DNA relative to E. coli PriB, suggests that the surface of this PriB homolog might have been adapted for a purpose other than binding ssDNA. Furthermore, it raises the important question of whether N. gonorrhoeae PriB can stimulate its cognate PriA’s helicase

Linsitinib cost activity, since in E. coli this stimulatory effect depends on PriB’s strong ssDNA-binding activity [7]. Figure 2 DNA binding activity of N. gonorrhoeae PriB. PriB was serially diluted and incubated with 1 nM fluorescein-labeled ssDNA (squares), dsDNA (closed diamonds), 3′ Overhang (circles), or Fork 2 (triangles). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. PriA helicase activity is limited to short stretches of duplex DNA To test the functional consequences of N. gonorrhoeae PriB’s weak DNA binding activity, we first had to examine N. gonorrhoeae PriA’s helicase activity. We used the partial duplex and forked DNA structures shown in Table 1 as substrates based on extensive XMU-MP-1 concentration studies of substrate preference

and helicase activity of E. coli PriA [22, 28, 29]. For each of these substrates, the fluorescein-labeled strand represents the nascent lagging strand arm, and the degree of duplex DNA unwinding of the fluorescein-labeled strand was determined using fluorescence polarization spectroscopy. For these experiments, the DNA substrates were incubated with PriA and ATP for 10 min at 37°C, the reactions were terminated by addition of SDS, and the fluorescence polarization of nearly the samples was measured. The degree of unwinding was determined by comparing the fluorescence polarization of the samples to that of the DNA substrate incubated in buffer alone (fully intact DNA substrate) and to the samples heated briefly to 95°C and fast-cooled back to 25°C (fully denatured DNA substrate). This allowed us to measure the fraction of each DNA substrate that is unwound by various concentrations of PriA. Of the DNA substrates examined, PriA shows greatest unwinding activity on forked DNA substrates with relatively short duplex lagging strand arms. Levels of maximal unwinding are approximately 83% for Fork 1 (15 bp lagging strand arm), 70% for Fork 2 (25 bp lagging strand arm), and 42% for Fork 3 (40 bp lagging strand arm) (Figure 3).