Our recent studies of the initiation process in DNA replication have focused on the cloning, characterization, and sequence determination of the bacterial origin (oriC) from six bacteria all of which function as origins in Escherichia coli. These studies have resulted in an oriC consensus sequence of conserved regions and required nucleotides. The consensus sequence demonstrates that oriC is the most complex regulatory site found in procaryotes. In this project, oriC structure will be related to its function in initiation and to regulation of the initiation process, by construction of mutant E. coli origins having known changes at specific positions. Oligodeoxynucleotide mutagenesis strategy will be dictated by consensus sequence information, predicted secondary structure, and protein/RNA binding and RNA transcription studies. Function will be related to specific structural changes by assaying replication properties of the mutant origins, and by assaying the effects of second site """"""""suppression"""""""" mutations.
The Specific Aims are: 1) Use oligodeoxynucleotide mutagenesis in specific regions of oriC where function depends: (a) on specific nucleotide sequence but not on secondary structure (binding sites); (b) on specific nucleotide sequence and on secondary structure (intrastrand helical stem regions); and (c) on length of the region, but not on its nucleotide sequence (nonconserved spacer regions separating binding sites). 2) Assay quantitatively the ability of mutant origin (a) to function as an origin (copy number, stability, incompatibility assays and (b) to function in the in vitro oriC initiation system. 3) Isolate and characterize origins from bacteria which do not methylate their GATC sites. These may be very different structurally and functionally from those that function in E. coli. The primary target in nearly all postulated mechanisms for transformation of a cell into a cancer cell is the cellular DNA. Loss of regulation of DNA replication and DNA repair leads to many diseases, including cancer, and regulation occurs mainly in the initiation process, both in eucaryotes and in procaryotes. This study of how the structure of the initiation site (origin of replication) is related to its function is thus directly relevant to the health sciences, and particularly to the cancer problem.
| Enns, R E; Garland, A M; Smith, D W (1986) M13-oriC cloning vehicles: use for amplification of high copy lethal (HCL) genetic elements. Plasmid 15:147-55 |
