Jon M. Kaguni Research Interests
Chromosomal replication in all organisms is an event that is tightly coupled to cell growth. This process can be separated into stages of initiation of DNA replication, progression of replication forks, termination of chromosomal DNA synthesis and segregation of daughter chromosomes into respective cells. Studies indicate that regulation of DNA replication occurs during the initiation of a cycle of chromosomal replication.
Of over 20 different genes that are required for replication of the Escherichia coli chromosome, the dnaA gene is uniquely required to initiate this process. My laboratory has focussed on the role of the dnaA gene product with the long range objectives of understanding biochemically the initiation of chromosomal replication, and its regulation. This protein is a sequence-specific DNA binding protein that binds to sites in the chromosomal origin of E. coli and in promoter regions of genes it regulates. One major approach has been to study the biochemical functions of DnaA protein in the step of initiation of DNA replication. The second is to isolate and characterize novel mutant forms of DnaA protein defective in replication. The third is to correlate structural domains of DnaA protein to its various functions.
From these approaches, we recently characterized a collection of dnaA mutants by genetic and biochemical methods that identify four functional domains. One domain near the N-terminus appears to be involved in interaction with DnaB protein, the replicative helicase responsible for progressive replication fork movement. A model has been proposed involving the DnaA-mediated oriented binding of DnaB helicase at the step of initiation so that it can function properly as a DNA helicase. Additional domains are involved in DNA binding, ATP binding, and in replication of pSC101. The latter is a plasmid of E. coli that requires DnaA protein for its replication. Other studies indicate that DnaA protein binds to four sites in the E. coli chromosomal origin in an ordered and sequential manner. Binding of DnaA protein to the last site, DnaA box R3, is critical in observing replication from the chromosomal origin both in vivo and in vitro. This event may be a key regulatory step in the initiation process. Finally, we have characterized biochemically a mutant form of DnaA protein with a substitution at alanine 184 with valine. Whereas wild type DnaA protein binds ATP with high affinity, this mutant protein binds ATP weakly, and requires activation by DnaK and GrpE heat shock proteins to observe ATP binding and replication activity. The impaired ability of DnaA protein to bind ATP results in a defect in the proper timing of DNA replication relative to the bacterial cell cycle. Control of ATP binding to regulate the replication activity of DnaA protein is apparently important in the proper timing of DNA replication in E. coli.