DNA replication, in bacteria, is initiated by a specific origin binding protein that recruits helicase assembly proteins and the replicative helicase. The helicase, once assembled on DNA, provides an interaction site for primase, the enzyme that generates RNA primers for DNA synthesis. The replicative helicase also plays a role in recruiting the cellular replicase, the DNA polymerase III holoenzyme that has the processivity to synthesize the entire chromosome without dissociation. In spite of this potential, most replicases encounter damage, resulting in replication fork destruction. This damage can be counteracted by a special origin independent replication restart apparatus that can reassemble replication forks. Altogether, these replicative reactions employ at least 20 different essential proteins. These protein targets and the essential interactions that occur between them provide attractive targets for the development of antibacterials, and will also serve as an ideal system for developing chemical genetic approaches to perturb the various interactions and reaction stages. We propose to develop robust HTS screening assays as well as appropriate specificity assays and counterscreens to enable the discovery of small molecule inhibitors that have the potential to be developed into antibacterials and step-specific perturbants of DNA replication pathways. These systems will be developed using model Gram (-) and Gram (+) organisms, E. coli and B. subtilis. These organisms are closely related to most common human pathogens and the biodefense category A organisms, Yersinia pestis and Bacillus anthracis, respectively. ? ?
| Dallmann, H Garry; Fackelmayer, Oliver J; Tomer, Guy et al. (2010) Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential. Biochemistry 49:2551-62 |
