We thank all the other members of the team for helpful discussions. Funding Statement This work was supported by: Grant 09-063428 from The Danish Medical Research Council, Grant R32-A2889 from The Lundbeck Foundation, Grant R144-A from The Novo Nordic Foundation, Danish Center for Antibiotic Research and Development (DanCARD) financed by The Danish Council for Strategic Research. the putative target (DnaN) which resulted in resistance. The minimum inhibitory concentration was 50 g/ml for cells. These compounds may serve as lead candidates for future development into novel classes of antibiotics as well as provide information on the function of the replication process. Introduction In recent years, many bacterial pathogens have become resistant or insensitive to most of the currently available antibiotics. As a consequence, infections caused by drug-resistant bacteria, including the Gram-positive methicillin-resistant (MRSA) and vancomycin-resistant (VRE) are associated with increased morbidity, mortality and health-care costs. The resistance problem has traditionally been addressed by development of semi-synthetic penicillins and the introduction into clinical use of novel antibiotic classes. This development peaked in the 1960s, and only two new classes of antibiotics, the oxazolidinones and daptomycin [1], [2], have been marketed within the last 30 years. In order to address the limited treatment options for Gadoxetate Disodium several bacterial infections it is important that the development of antimicrobials continue and Gadoxetate Disodium include both new targets for intervention as well as new classes of inhibitors. Chromosome duplication is an essential process in all living organisms and the multienzyme machinery that replicates bacterial DNA represents one such underexploited target. In bacteria the replication process is carried out by highly conserved proteins, which deviate from their eukaryotic counterparts in structure and sequence (reviewed by [3]). Compounds that target bacterial DNA replication are therefore expected to have a high therapeutic index. Most of our current knowledge on bacterial chromosome replication comes from studies of replication origin, and flanking the DUE (Duplex Unwinding Element) region is essential for helicase loading, and is stimulated by the formation of a second DnaA sub-complex in the right half of DNA wrapped around it. Binding of IHF immediately upstream of the DUE flanking R1 DnaA-box introduces a 160 bend in the DNA reversing the orientation of the DNA helical axis and assist in melting the DUE region. One of the exposed single-stranded DUE regions is fixed by binding the existing DnaA-ATP helix while the other strand is exposed for DnaC assisted DnaB helicase loading by the DnaA molecule bound to the R1 box. Further opening of the duplex allows for loading of the second helicase by one or more N-terminal domains of the DnaA-ATP Gadoxetate Disodium filament [5]. Although promoted by formation of a DnaA oligomer on with a couple of notable exceptions. The helicase (called DnaC) is loaded by the DnaI helicase loader assisted by the DnaB and DnaD proteins [9] and two different replicative polymerases are used. The DnaE which is homologous to the PolIII only extends RNA primers initially and hands them off to PolC which is responsible for the processive synthesis (reviewed in [10]). A third difference was recently revealed. Primer hand off in was achieved by manipulation of protein splicing (SICLOPPS; split intein-mediated circular ligation of peptides and proteins) which utilizes the DnaE split intein of sp. PCC6803 [23], [25]C[28]. This method coupled to reverse bacterial two-hybrid system allowed us Gadoxetate Disodium to select peptides that were able to decrease protein-protein interactions of selected pairs of replication proteins. Peptides targeting DnaN-DnaN interaction were further characterized with respect to target specificity and activity. A similar approach has earlier been used to identify cyclic peptides that inhibit the ribonucleotide reductase by hampering association between NrdA and NrdB subunits [29]. Results Protein-protein interactions in the replicative DNA polymerase and its loaders have been extensively characterized by biochemical and biophysical approaches. In order to demonstrate interactions between replication proteins in we used the bacterial two hybrid (BTH) system developed by Karimova et al. [30]. This system is based on interaction-mediated reconstruction of adenylate cyclase activity in the adenylate Rabbit Polyclonal to Caspase 14 (p10, Cleaved-Lys222) cyclase deficient strain BTH101 (Table 1). In this system the Cya protein of is split into two domains (T18 and T25) resulting in loss of activity. If T18 and T25 are fused to interacting polypeptides the two Cya domains will be brought into proximity of each other Gadoxetate Disodium to create a Cya+ phenotype. This results in cAMP production and consequently in activation of cAMP-CAP regulated promoters (e.g the promoter). Table 1 Bacterial strains. ::RN450 [60] 8325-4 transcriptional fusion [62] Open in a separate window We fused.
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