The overall objective of this project is to investigate the duplication process of E. coli cells with particular regard to the initiation and replication of chromosomal DNA, the control and process of cel division, and the coordination between DNA replication and the celllar division cycle. Since the mechanisms which determines the frequency of initiation of chromosome replication is a key regulatory step in the cell cycle, studies on the control of initiation from the chromosomal origin, oriC, are emphasized. The research plan is divided into two basic areas: physiological studies aimed at identifying the elements which coordinate chromosome replication with the cellular growth cycle in vivo, and in vitro studies on the molecular biology of oricC-directed DNA replication. Many of the projects in vivo and in vitro will employ a variety of minichromosomes, i.e., small DNA molecules which replicate from a resident copy of oricC. The specific projects are: 1) To determine if the timing of initiation of chromosome replication in E. coli could be regulated positively by components of the complex of activities required for initiation. Recent findings have led to the development of an experimental strategy which will enable this question to be answered. It will be determined, for instance, whether the apparent absence of reutilization of initiation complexes for initiation at oricC is the basis for the regulatory system in vivo. 2) To compare replication properties of plasmid-driven chromosomes with oricC-driven chromosomes to determine whether the oricC origin-complex possesses unique properties of a growth coupled initiation frequency regulator. 3) To characterize the kinetics of synthesis, cellular content, stability and segretion of individual proteins known to be associated with initiation and/or replication. Experiments are planned to relate these findings to the prediced properties of positive regulatory elements. The second series of experiments involves comparative analyses of minichromosome DNA replication in vitro and in vivo. The intermediates through which mninchromosomes progess during replication will be determined, along with an analysis of topological requirements for replication. The molecular species which form early during replication in vitro and in vivo will be examined in detail.
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