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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Dearolf, Charles R
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Duke University
Schools of Medicine
United States
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Long, David T; Kreuzer, Kenneth N (2009) Fork regression is an active helicase-driven pathway in bacteriophage T4. EMBO Rep 10:394-9
Pohlhaus, Jennifer Reineke; Kreuzer, Kenneth N (2006) Formation and processing of stalled replication forks--utility of two-dimensional agarose gels. Methods Enzymol 409:477-93
Dudas, Kathleen C; Kreuzer, Kenneth N (2005) Bacteriophage T4 helicase loader protein gp59 functions as gatekeeper in origin-dependent replication in vivo. J Biol Chem 280:21561-9
Kreuzer, Kenneth N (2005) Interplay between DNA replication and recombination in prokaryotes. Annu Rev Microbiol 59:43-67
Stohr, Bradley A; Kreuzer, Kenneth N (2002) Coordination of DNA ends during double-strand-break repair in bacteriophage T4. Genetics 162:1019-30
Jones, C E; Mueser, T C; Dudas, K C et al. (2001) Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: a versatile couple with roles in replication and recombination. Proc Natl Acad Sci U S A 98:8312-8
Dudas, K C; Kreuzer, K N (2001) UvsW protein regulates bacteriophage T4 origin-dependent replication by unwinding R-loops. Mol Cell Biol 21:2706-15
Doan, P L; Belanger, K G; Kreuzer, K N (2001) Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not. Genetics 157:1077-87
George, J W; Stohr, B A; Tomso, D J et al. (2001) The tight linkage between DNA replication and double-strand break repair in bacteriophage T4. Proc Natl Acad Sci U S A 98:8290-7
Nossal, N G; Dudas, K C; Kreuzer, K N (2001) Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro. Mol Cell 7:31-41

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