DNA REPLICATION PROTEINS OF GRAM POSITIVE BACTERIA AND THEIR USE TO SCREEN FOR CHEMICAL INHIBITORS

摘要

The duplex DNA of chromosomes is replicated in a multicomponent process. A helicase unwinds the DNA, a replicase synthesizes new DNA, and primase repeatedly synthesizes new primed starts on the lagging strand. The present invention is directed to the genes from Gram positive bacterium encoding these proteins, and their characterization. Replicases are highly efficient polymerases. There are several mechanisms by which a replicase can achieve high processivity. The invention determines that the replicase of Staphylococcus operates as a 3 component system in which a clamp loader enzyme assembles a sliding clamp protein onto DNA. The sliding clamp then binds the DNA polymerase III holoenzyme making it highly efficient. The invention identifies two DNA polymerase III enzymes in Gram positive bacterium, each of which operate with the clamp and clamp loader, to extend a single primed site around a long (over 5kb) ssDNA template. These replication proteins can be utilized in a variety of assays to screen chemical compound libraries for an antibiotic compound.