The Completion of DNA Replication

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The mechanism by which chromosome replication is accurately completed is not well understood. To maintain genomic integrity during replication, cells must have an ability to recognize replicated regions, limit the ongoing replication process, and join converging daughter strands at the precise point at which the DNA sequence has doubled. Despite these complexities, this process occurs with remarkable efficiency (occurring thousands of times per division in human cells) and is fundamental to the accurate duplication of the genome. It appears to be a common point of deregulation in many cancer cells and is deliberately targeted for inactivation by viruses to induce the lytic phase of replication (wherein the virus multiplies rapidly). This project will determine how cells sense replicated regions and accurately complete the proess at the precise doubling point in the model organisms, Escherichia coli and Saccharomyces cerevisiae. Students trained during this project will be qualified for careers in research, biotechnology and medicine in both academia and industry. The project will also expand the number of undergraduates who participate in research experiences at research centers in the region and expand the math and science opportunities for K-8 students in the public school system.Completion of replication in E. coli appears to involve a transient over-replication of the region where forks converge before the excess regions are incised, resected, and joined. By monitoring the copy number of DNA sequences where completion occurs in E. coli, the research will identify mutants that have an impaired ability to accurately complete replication. Using plasmid minichromosomes, a combination of 2D and 1D agarose gel analysis will then be used to identify the structural intermediates that arise during the completion of replication in both wild type and mutant cells. Finally, the research will determine if the completion mechanism is conserved in eukaryotes, by examining the homologous mutants in yeast to identify whether similar abnormalities on eukaryotic chromosomes are observed where replication forks converge.