DNA replication in phage T4 is initiated by two distinct modes, both of which require particular DNA structures. Origin replication initiates from R-loops, while recombination-dependent replication (RDR) initiates from D-loops. A topological assay will be used to measure R-loop formation at T4 origins in vivo. The results should clarify the mechanism of R-loop formation and test the model that UvsW protein unwinds R-loops to repress T4 origins at late times. The detailed roles of replication proteins and origin RNA will also be analyzed, for example asking whether translation of the origin RNA reduces replication, determining whether gp59 acts as a """"""""molecular gatekeeper"""""""" of the replication apparatus in vivo, and testing whether T4 topoisomerase is the major fork swivel and decatenase. A new area of investigation will attempt the isolation of replication origins active in constitutive stable DNA replication in uninfected E. coli, which is also proposed to operate by an R-loop mechanism. Studies of T4 RDR have uncovered a tight linkage between replication, recombination and dsb repair. The products and requirements for dsb repair will be analyzed in plasmids and in phage chromosomes to distinguish between three different models. In addition, dsb-promoted DNA replication will be analyzed within the context of the phage genome. Using phage T4 recombination hotspots that are only active on damaged DNA, the process of replication fork blockage at damage and fork restart will also be approached. Fork restart is likely a specialized pathway of RDR. The proposed studies have significant health relatedness because the T4 system continues to provide important lessons relevant to human cell replication, recombination and repair. These processes are critical in many medically relevant areas, such as early development, generation of antibody diversity, response to both carcinogenic and anticancer agents, and maintenance of chromosomes in proliferating tumor cells.
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