Replication Associated Genetic Rearrangements

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In recent years evidence has accumulated that replication forks frequently arrest and require repair to be restarted. Recombinational processes therefore underpin replication to permit replication of the entire chromosome. Our study of genetic rearrangements that are induced by defects in the DnaB replicative helicase of E. coil has revealed a requirement for the RadA/Sms protein. In several recombination and DNA repair assays, the radA gene plays a role redundant with ruvABC and recG, genes that encode Holliday junction processing proteins. RadA therefore most likely acts to stabilize or process recombination intermediates. RadA is a ubiquitous eubaeterial protein that shares sequence similarity to the ReeA/Rad51Dmc 1 strand exchange factors and to Lon protease. Our first specific aim is to characterize the biochemical properties of purified RadA protein tbr DNA binding, strand exchange, protease and recombination intermediate cleavage activities. The elucidation of its biochemical properties should reveal how RadA acts in recombination and DNA repair. Three areas of replication fork repair in bacteria remain incompletely understood: the replication checkpoint response, the influence of cellular localization of the chromosome and proteins on repair and the orchestration of assembly and disassembly reactions during repair. Our second aim seeks to clarify these arcas by genetic analysis of replication fork repair in E. coli. Sensitivity to hydroxyurea, an inhibitor of ribonucleotide reductase, has been used to identify fork repair and replication checkpoint mutants in yeast. Our pilot screen for HU-sensitive mutants of E. coli has revealed a signal transduction protein that may regulate a cell division checkpoint and further characterization of this mutant is proposed to test this hypothesis. Several interesting mutants with recA-synthetic DNA damage or viability phenotypes have been isolated and will be characterized. Both traditional and genomics-based mutant analyses are proposed. Proteins known to control replication initiation and chromosome localization will also be investigated for effects on fork repair. Our third aim is to perform microarray analysis of E. coli genomic transcription in response to replication fork arrest or damage. These may reveal novel functions involved in the cellular response to replication fork arrest. This aim complements the second by identifying candidate genes that may control tolerance or repair of fork damage. Furthermore, mutants identified in the second aim can be screened for effects on global gene expression after conditions of replication arrest.