Structural and Biochemical Studies of the DNA Replication Initiation Mechanism of the Second Chromosome of \u3ci\u3eVibrio cholerae\u3c/i\u3e

Transmission of genetic information through DNA replication is one of the key processes for any living organism. Despite the extensive effort put into studies of the mechanism of DNA replication, the understanding of the process on the molecular level is still incomplete. Specifically the molecular details of the very first events of DNA replication initiation are not sufficiently understood. The majority of bacteria possess a single circular chromosome, and in order to initiate DNA replication these organisms utilize a conserved system, consisting of a specific DNA sequence - replication origin, called oriC, and replication initiator protein DnaA. However, bacteria with multipartite genomes evolved distinct systems to initiate replication of secondary chromosomes. In Vibrio cholerae, and in related species, secondary chromosome replication requires specific origin of replication, OriCII, and unique initiator protein, RctB, which shares no sequence similarity with other initiator proteins. Previous structural studies of bacterial DNA replication initiation were conducted in two kinds of systems: bacterial primary chromosomes and bacterial plasmids; these studies have been challenging, and there are still many opened questions to date. No prior structural work was done in order to elucidate mechanism of DNA replication initiation in secondary bacterial chromosomes, such as DNA replication initiation conducted by RctB-oriCII system. The work described in this dissertation is dedicated to structural studies of RctB-oriCII system. The structures of all four RctB domains were solved using X-ray crystallography and biophysical and biochemical analyses of RctB-DNA complexes were conducted. This work produced first structural view for the secondary chromosomal DNA replication initiation machinery and laid out a strong foundation for future structural studies of RctB-OriCII complexes. We demonstrate that RctB consists of four domains. The structure of its central two domains resembles that of several plasmid replication initiators. RctB contains at least three DNA binding winged helix turn helix motifs, and mutations within any of these severely compromise biological activity. In the structure, RctB adopts a head-to-head dimeric configuration that likely reflects the arrangement in solution. Therefore, major structural reorganization must accompany complex formation on the head-to-tail array of binding sites in oriCII. These findings support the hypothesis that the second Vibrionaceae chromosome arose from an ancestral plasmid, and that RctB may have evolved additional regulatory features. The structural and biophysical data obtained in the course of this work allowed us to develop a model of the macromolecular complex that several molecules of RctB form on replication origin of the secondary Vibrio cholerae chromosome