Genomic Mapping of Replication Origins in Higher Eukaryotes by Okazaki Analysis

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DESCRIPTION (provided by applicant): DNA replication is one of the most fundamental biological processes. Starting from multiple sites (replication origins) encoded mostly in non-coding regions of the genome, the completion of duplication of the entire genome is highly regulated and essential for the survival of any species. Despite the very basic nature of this process, less than a few dozen of replication origins in metazoan cells have been characterized to date. Our knowledge on the number of origins, the sites of replication termination and the arrangement of replicons in the genome remains very limited. Here, we propose to develop a versatile, genomic scale mapping method based on strand analysis of Okazaki fragments using short, sequence specific tags generated by lype Us en/ymes. Using the budding yeast as a tractable model system, the detailed procedures of this approach will be validated and optimized. With this method, not only are the replication origins mapped to a high resolution in parallel, but also the fork termination sites that are required to define each individual replicons in the genome. To date, it has not been possible to map the sites of fork termination directly on a large scale. To apply this approach to the much larger human genome containing considerable amount of repeat sequences, the later half of this application is focused on the development of effective protocols to isolate Okazaki fragments on a select region of the genome with the majority of repeat sequences removed. This reduced complexity is required to achieve unique matching of tags of limited length to target sequences. For this purpose, a portion of the sequences selected by the ENCODE project will be used. When these objectives are accomplished, the feasibility of this approach will have been fully demonstrated, and large scale mapping of the human genome, as well as other metazoan genomes, can be initiated with confidence. Among many possible utilities of such comprehensive datasets is the comparative examination of various metazoan genomes that will most certainly reveal the organization and conservation of replicons in the chromosomal context, further elucidating the evolution of one of the most important functional signals embedded in noncoding regions of the genome. It is probable that when the number of known origins increases, consensus sequence motifs or signatures may also be discovered, which can play critical roles in defining and regulating the function of replication origins.