DNA replication in bacteriophage T4 is initiated by two different modes, dependent initially on replication origins and then progressing to a mechanism dependent on recombination proteins. The proposed studies in this application are directed toward understanding the mechanisms of these two modes and the nature of the transition between them. The studies on origin-dependent replication focus on the role of a persistent RNA-DNA hybrid (R loop) that were recently detected at a t$ replication origin in the applicant's laboratory. Proposed experiments will seek corroborating evidence for the existence of the R loop in vivo by at least one additional method, and will explore the requirements for formation of R loops in vivo. Parallel experiments will analyze the formation of the loop in vitro and test a specific model of R loop formation. The transition from origin-dependent to recombination-dependent replication involves the inactivation of phage replication origins at late times by the phage- encoded UvsW protein. Recent evidence suggests that UvsW may be an RNA-DNA helicase that removes the persistent hybrid from the origin. This model will be tested both in vivo and in vitro. The recombination-dependent mode of DNA replication, which predominates at late times of the T4 multiplication cycle, is very closely related to the process of recombinational repair of DNA. Recent experiments indicate that double-strand breaks are repaired by a mechanism that is essentially identical to recombination-dependent replication, leading the applicant to propose an 'extensive chromosome replication' (ECR) model for double strand break repair of DNA. Several aspects of the ECR model will be tested. The detailed steps of the coupled repair/replication reaction, particularly the early steps involved in activating double-stranded ends and the late steps of Holiday junction migration and resolution will also be investigated. The applicant stresses that the proposed studies have significant health relatedness because the T4 replication system has striking similarities to human cell DNA replication, and because double strand break repair is important in processes such as generation of antibody diversity, response to carcinogenic agents, and perhaps maintenance of chromosomes in certain proliferating tumor cells.
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