The long-term objective of this research program is to understand the bacterial replication cycle of Escherichia coli and how it is coordinated with the cell division cycle. Topics of particular interest are the structure of the bacterial chromosome and how this changes during the replication cycle, the mechanism by which daughter chromosomes are decatenated and then partitioned to daughter cells, and how completion of the replication cycle is related to subsequent cell division. We are currently studying the terminus region of the chromosome and have identified several loci that are important for the normal replication cycle and other chromosomal events. This proposal will continue those studies. Specifically, it is proposed to do the following: 1) Determine how the tus gene, whose product is required for inhibition of replication at terminator sites T1-T4, in controlled. We will determine which promotors are used for tus expression, whether expression is autoregulated, whether other factors are also involved in tus expression, and whether its expression changes during the replication cycle. 2) Determine which other proteins, besides Tus, are involved in the inhibition of replication. A mutant search will be conducted that will identify other factors required for inhibition of replication at terminator sites and proteins of the replication apparatus with which Tus interacts. 3) Isolate a set of mutants that exhibit temperature sensitive inhibition. These will be primarily tus mutants, and they will be used as the basis for constructing a plasmid system in which inhibition can be controlled by temperature shifts. This will be used to characterize the events that occur before, during, and after the meeting of replication forks in an artificial terminus. Information obtained form these studies will then be used for studies at the chromosome level. 4) Characterize the dif locus. dif is located in the terminus, is cis- acting, and its removal leads to SOS induction. We will characterize if dif is required at a particular time during the replication cycle, what is the inducing signal for SOS, whether dif can function at other locations in the chromosome, and test the effect of dif on interchromosomal and intrachromosomal recombination.
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