Maximal unwinding activity is approximately 19% for this substrate, suggesting that the partial duplex DNA lacks structural elements required for efficient PriA binding and unwinding (Figure 3). This has been observed for E. coli PriA helicase as well [7, 28]. Overall, these results demonstrate
that N. gonorrhoeae PriA helicase activity is limited to relatively short stretches of duplex DNA, akin to its E. coli counterpart. Figure 3 Helicase activity of N. gonorrhoeae PriA. PriA-catalyzed duplex DNA unwinding was examined using 1 nM Fork 1 (15 bp lagging strand arm, diamonds), Fork 2 (25 bp lagging strand arm, triangles), Fork 3 (40 bp lagging strand arm, squares), or 3′ Overhang buy BIIB057 (25 bp partial duplex, circles). Measurements are reported in triplicate
and error bars represent one standard deviation of the mean. Comparison of the helicase activity of N. gonorrhoeae PriA that we measured in this study with the previously reported helicase activity of E. coli PriA at the same concentrations and on similar DNA substrates reveals that the two PriA homologs follow the same trend with respect to the dependence of their DNA unwinding activity on the length of the duplex arm of the DNA substrate (Table 3). There are some differences in the degree of DNA unwinding catalyzed by N. gonorrhoeae PriA that we measured in this study compared with the helicase activity previously reported for E. coli PriA. For example, E. coli PriA helicase shows slightly elevated DNA unwinding activity on the 25 bp fork structure compared to N. gonorrhoeae PriA KU55933 mw (Table 3). Whether this represents natural biological variation between the two PriA homologs or differences arising from work involving separate investigators is uncertain. Table 3 Comparison of helicase activity of E. coli PriA and N. gonorrhoeae PriA. DNA Substrate E. coli PriA1 % DNA
Unwound N. gonorrhoeae Vildagliptin PriA2 % DNA Unwound 25 bp fork 83 ± 3 61 ± 6 40 bp fork 28 ± 8 37 ± 7 25 bp partial duplex 23 ± 2 17 ± 4 1Cadman et al. J Biol Chem 2005, 280(48):39693-39700. 2This study. In this study, the 25 bp fork substrate is Fork 2, the 40 bp fork substrate is Fork 3, and the 25 bp partial duplex substrate is 3′ Overhang. The helicase activity for each PriA homolog is the mean percent of DNA unwound by 5 nM PriA on 1 nM DNA substrate and in the PARP inhibitor absence of its cognate PriB. Mean values from Cadman et al. are derived from two independent experiments, and mean values from this study are derived from three independent experiments. Associated uncertainty values are one standard deviation of the mean. PriB stimulates PriA’s helicase activity on long regions of duplex DNA To determine if N. gonorrhoeae PriB stimulates the helicase activity of its cognate PriA, we examined PriA helicase activity on a forked DNA substrate with a 40 bp lagging strand arm (Fork 3) in the presence and absence of PriB.