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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032968-10
Application #
3282237
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-01-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Hendricks, E C; Szerlong, H; Hill, T et al. (2000) Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli. Mol Microbiol 36:973-81
Hojgaard, A; Szerlong, H; Tabor, C et al. (1999) Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV. Mol Microbiol 33:1027-36
Steiner, W W; Kuempel, P L (1998) Sister chromatid exchange frequencies in Escherichia coli analyzed by recombination at the dif resolvase site. J Bacteriol 180:6269-75
Steiner, W W; Kuempel, P L (1998) Cell division is required for resolution of dimer chromosomes at the dif locus of Escherichia coli. Mol Microbiol 27:257-68
Kuempel, P; Hogaard, A; Nielsen, M et al. (1996) Use of a transposon (Tndif) to obtain suppressing and nonsuppressing insertions of the dif resolvase site of Escherichia coli. Genes Dev 10:1162-71
Tecklenburg, M; Naumer, A; Nagappan, O et al. (1995) The dif resolvase locus of the Escherichia coli chromosome can be replaced by a 33-bp sequence, but function depends on location. Proc Natl Acad Sci U S A 92:1352-6
Gottlieb, P A; Wu, S; Zhang, X et al. (1992) Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction. J Biol Chem 267:7434-43
Roecklein, B A; Kuempel, P L (1992) In vivo characterization of tus gene expression in Escherichia coli. Mol Microbiol 6:1655-61
Kuempel, P L; Henson, J M; Dircks, L et al. (1991) dif, a recA-independent recombination site in the terminus region of the chromosome of Escherichia coli. New Biol 3:799-811
Hill, T M; Tecklenburg, M L; Pelletier, A J et al. (1989) tus, the trans-acting gene required for termination of DNA replication in Escherichia coli, encodes a DNA-binding protein. Proc Natl Acad Sci U S A 86:1593-7

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