Studies of DNA replication restart pathways in diverse bacteria such as E. coli and N. gonorrhoeae have revealed species differences in the composition of the DNA replication restart primosome and in the AZD6244 clinical trial functions of the individual primosome proteins. For example, N. gonorrhoeae lacks a recognizable homolog of dnaT in its genome, suggesting that the N. gonorrhoeae PriA-PriB pathway might be significantly different from the E. coli PriA-PriB-DnaT pathway. Furthermore, physical interactions between primosome components show variation in their individual binary affinities: the

physical interaction between PriA and PriB is rather weak

in E. coli, but relatively strong in N. gonorrhoeae, and the physical interaction between PriB and ssDNA is strong in E. coli, but relatively weak in N. gonorrhoeae [8, 17, 18]. Thus, the affinities of binary interactions between primosome components are reversed between the two species. Since the ssDNA-binding activity of PriB is important for PriB-stimulation of PriA’s HDAC inhibitor helicase activity in E. coli [7], there might be significant functional consequences for the variation in affinities of physical interactions within the N. gonorrhoeae PriA-PriB primosome. In this study, we investigated the

functional consequences of the affinity reversal phenomenon by examining the helicase activity of N. gonorrhoeae check details PriA, and we determined how PriA-catalyzed ATP hydrolysis and DNA unwinding are affected by N. gonorrhoeae PriB. Results DNA binding by PriA, but not PriB, is structure-specific We used fluorescence polarization spectroscopy to examine the physical interaction between N. gonorrhoeae PriA and a variety of DNA structures that mafosfamide were constructed by annealing fluorescein-labeled synthetic DNA oligonucleotides. The DNA structures include ssDNA, a partial duplex DNA with a 3′ ssDNA overhang, and a forked DNA structure with fully duplex leading and lagging strand arms (Table 1). The presence of a fluorescein tag on the DNAs allowed us to measure PriA binding to the DNA due to the increase in fluorescence polarization of the PriA:DNA complex relative to the unbound DNA. PriA protein was serially diluted and incubated with 1 nM fluorescein-labeled DNA and the fluorescence polarization was measured. Apparent dissociation constants were obtained by determining the concentration of PriA needed to achieve 50% binding to each of the various DNA substrates. Table 1 DNA substrates.