The decision to advance from G1 into the replicative (S) phase is among the most highly regulated in the cell cycle. Stringent control over entry into S-phase is crucial since DNA replication under inappropriate condiitons can result in chromosome breakage and lead to either cell death or the emergence of genetic variant that accelerate cancer progression. An explosion of knowledge over the past few years has generated a detailed picture of how the cell senses and responds to signals elicited by DNA damage and various growth or arrest cues. In contrast to the wealth of information about these signaling pathways, there is virtually no understanding of the mechanisms by which this information is transduced to the DNA duplex, the ultimate response element in the decision to enter S- phase. Progress in understanding how information is transmitted from the cell cycle machinery to the DNA duplex requires detailed information about structure-function relationships at replication origins. The central goals of this proposal are to define the molecular structures required for initiation of DNA replication in mammalian cells, and to determine whether cell cycle regulated changes correlated with their function. These goals will be achieved using two products of the last grant cycle; 1) new genetic approaches involving the site specific recombinase FLP for dissecting initiation regions in complex genomes, and 2) molecular clones containing initiation regions identified in mammalian chromosomes. FLP recombinase will allow a genetic analysis of whether """"""""licensing"""""""" of candidate origins by cell cycle regulated modifications is required for origin function. Wild type and mutated origins will be inserted into specified sites in the genome using the FLP recombinase, and biochemical assays will be employed to compare their abilities to initiate replication. FLP -mediated excision will be used to determine whether the putative origins enable extrachromosomal, autonomous replication. Function within and outside the chromosome fulfills the genetic criteria for an origin. Analysis of an initiation region located in the developmentally regulated human b-globin locus will investigate potential links between transcription and replication control. We will interchange putative regulatory components to determine whether origins are organized in modular arrays like transcriptional promoters, and whether transcriptional regulatory elements are involved in origin function. Accomplishment of these goals using an integrated biochemical and genetic approach should elucidate the molecular structure of mammalian replication origins, and how initiation of DNA replication may be coupled to the cell cycle control machinery.
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