The primary objective of this project is to determine the function(s) of methylation of GATC sites in DNA replication initiation events at the bacterial origin (oriC). Our previous studies have shown that six naturally occurring E. coli-type bacterial origins each contain 10-14 GATC sites within the 245 bp minimal origin, and 8 of these are perfectly conserved. We have further shown that GATC methylation state in oriC is highly important in oriC ability to function in vivo and in vitro as a bacterial origin. This project will distinguish between 1) direct effects of GATC methylation on origin function (binding of trans-acting initiation molecules; oriC required secondary structure); 2) regulatory function (time required between initiation events); and 3) indirect effects (promoters of genes whose products are involved in initiation).
The Specific Aims are: 1) Determine GATC methylation state of specific sites using single origin and replicating oriC and Co1E1 plasmids, and using the chromosomal origin. Use GATC enzymes (Sau3A, Dpnl, Mbol) to assay methylation patterns. Use both dam+ and dam- cells. Correlate results with non-replicating plasmid DNA. Use chromosomal oriC DNA isolated from dnaA, dnaB, and dnaC mutant cells synchronized before and during initiation. 2) Examine in vivo and in vitro initiation properties of totally nonmethylated oriC and ColEl origin plasmids isolated from yeast. 3) Isolate and characterize mutants in origin GATC sites for oriC and the ColEl origin. Obtain suppressor mutants of these initial mutants. 4) Isolate and characterize E. coli GATC methylation mutants. Deletion/insertion mutants of the dam gene, gene for the DNA adenine methylase, are viable and appear to exhibit residual GATC methylation ability, suggesting presence of a """"""""backup"""""""" methylation system. 5) Delineate the """"""""dam operon"""""""". Identify transcription events via hybridization and S1 nuclease mapping experiments. Identify gene products via protein expression in maxicells and via DNA sequencing. 6) Determine the extent and type of control of expression of the dnaA and dam genes by the DnaA and Dam proteins. Methylation events are implicated in both DNA repair and DNA replication in procaryotes, as well as in some gene expression, and undermethylation appears to be very important in gene expression in eucaryotes. Loss of regulation of DNA metabolic processes, particularly replication and repair, and of correctly regulated gene expression leads to many diseases, including several forms of cancer. Thus, this project is directly related to the health sciences.
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