The long-term objective of this project is to elucidate the molecular mechanisms used by phage T4 to initiate DNA synthesis, with particular emphasis on the relationships between replication, transcription and recombination. The three known modes of replication initiation in phage T4 are distinguished by their dependencies on transcription and recombination functions. Primary initiation, which will not be pursued in this proposal, is dependent on a direct involvement of RNA polymerase. Secondary initiation is independent of transcription but requires phage-encoded proteins that are involved in genetic recombination. Plasmids containing non-origin fragments of the T4 genome nonetheless replicate after T4 infection by a mechanism closely related to secondary initiation. This simplified in vivo model system will be used to explore the protein requirements and to study the mechanism of secondary initiation. Tertiary initiation is resistant to the RNA polymerase inhibitor refampicin, and yet a middle-mode promoter is an essential component of tertiary origins. In vivo experiments will focus on the roles of particular proteins and on the mechanisms of rolling circle production from tertiary origin plasmids. The role of RNA polymerase will be explored by the in vitro reconstitution of middle transcription from the tertiary origin promoters. Additional initiation proteins will be purified on the basis of specific binding to either the tertiary origin DNA or the middle- mode transcriptional apparatus. With at least some of the initiation proteins and suitable origin DNA substrates in hand, the in vitro reconstitution of tertiary initiation will then be pursued. The two known tertiary origins coincide with recombination hotspots, and secondary initiation requires several recombination proteins. Therefore, a direct analysis of recombination mechanisms will also be executed to complement the replication studies. The integration of recombinant plasmids into the phage genome provides a very sensitive assay for homologous recombination, and appears to occur by at least two pathways. The protein requirements for these pathways will be determined as a necessary step towards dissecting the mechanisms of recombination. Phage T4 appears to provide an ideal model system in which to explore the relationships between replication and recombination.
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