INITIATION OF DNA REPLICATION IN THE PHAGE T4 SYSTEM

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The long-term goals of this project are to elucidate the molecular mechanisms used by bacteriophage T4 to initiate DNA replication, and to understand the relationships between phage replication, recombination and transcription. As judged by striking similarities in the DNA metabolic machineries of T4 and eukaryotic cells, the phage T4 model system may be particularly relevant to studies of human chromosomal replication, recombination and repair, and thus to human health. Two well-characterized T4 replication origins contain a middle-mode promoter and a downstream segment of DNA that readily unwinds. The roles of the promoter and downstream region in initiation will be explored, using origin DNA mutations and T4 mutants that lack particular replication proteins. One interesting model is that the combination of a promoter and an unwinding region leads to a stable RNA-DNA hybrid in the unwinding region. This model will be directly tested in vitro, and the results could have important implications for both transcription and DNA replication. A new system of transposon mutagenesis will also be used to find additional genes involved in origin-dependent replication and in T4 recombinational processes. The mechanism of recombination-dependent T4 replication will be explored using a novel system in which DNA replication is triggered by site-specific double-strand breaks in the viral genome. Specific intermediates in recombination-dependent replication will be analyzed in wild type and various mutant infections, and the requirement for homologous DNA segments will be further explored. Direct studies of T4 recombination will also utilize site-specific double-strand breaks in the phage genome. A single-strand annealing model for genetic recombination has long been postulated for phage T4 and has recently emerged for recombination between repeated eukaryotic genes. This model will be tested by analyzing the recombination of DNA molecules that could provide complementary single-stranded regions adjacent to double-strand breaks. The mechanism of recombination at native T4 recombination hotspots will also be examined, particularly because these hotspots are triggered by the above-mentioned replication origins of the virus.