The asterisk denotes the position of the fluorescein

label. Numbers in parentheses denote the number of bases in the oligonucleotide. As expected based on studies find more of E. coli PriA DNA binding [5, 19–22], N. gonorrhoeae PriA binds each of the DNA structures that we tested (Figure 1). PriA binds the forked DNA structure (Fork 2) with the highest affinity of the DNA structures tested, resulting in an apparent dissociation constant of 134 ± 22 nM (Table 2). This DNA structure has fully duplex leading and lagging strand arms with no gap at the three-way junction, and a hydroxyl group exists at the 3′ end of the leading strand arm to provide contacts with the 3′ hydroxyl binding pocket of PriA’s DNA binding domain, assuming that this feature of the helicase has been conserved between the E. coli and N. gonorrhoeae homologs [23]. Figure 1 DNA binding activity of Trichostatin A N. gonorrhoeae PriA. PriA was serially diluted and incubated with 1 nM fluorescein-labeled ssDNA (squares), 3′ Overhang (circles), or Fork 2 (triangles). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. Table 2 Apparent dissociation constants for PriA:DNA and PriB:DNA

complexes. DNA Substrate PriA Kd,app, nM PriB Kd,app, nM ssDNA 307 ± 43 662 ± 37 dsDNA ND 640 ± 35 3′ Overhang 234 ± 62 628 ± 95 Fork 2 134 ± 22 690 ± 51 Apparent dissociation constants (Kd,app) are mean values derived from at least three independent experiments and associated uncertainty values are one standard deviation of the mean. ND: Not determined. The apparent dissociation constants for the partial duplex DNA with a 3′ ssDNA overhang and the ssDNA substrate are higher than that of the forked DNA substrate, with values of 234 ± 62 nM (3′ Overhang) and 307 ± 43 nM (ssDNA) (Table 2). While Sitaxentan we can not rule out the possibility that the differences in affinity are due to differences in the size of the DNA substrates, it is possible

that the partial duplex DNA and the ssDNA substrates lack structural elements that are needed to achieve the high affinity binding observed with the forked DNA substrate. Work from several laboratories has demonstrated that E. coli PriB is a ssDNA-binding protein [18, 24–27], and previous work from our laboratory has shown that N. gonorrhoeae PriB binds ssDNA, albeit with a significantly lower affinity than does the E. coli PriB homolog [17]. Despite this lower affinity, N. gonorrhoeae PriB has the structural hallmark of a ssDNA-binding protein [17], leading us to hypothesize that it would bind ssDNA and any DNA structures that contain ssDNA with higher affinity than duplex DNAs.