We are interested in control mechanisms operating during initiation of DNA replication and how they interface with control of growth. Specifically, we want to investigate the relationship of DNA replication, recombination, transcription and packaging in the life cycle of phage T4, which is well characterized genetically and biochemically. Like many other viruses, T4 uses at least two modes to initiate DNA replication. Our recent results have shown that the first mode is initiated de novo from origin sequence(s) and requires RNA polymerase for priming while the second mode initiates from recombinational intermediates independent of host RNA polymerase. We now want to address the following problems: 1. Determine """"""""functional domains"""""""" of a primary origin region between genes dda and 56, and of a secondary origin in the uvsy-w-dar region by sequencing and mutating these regions, and studying their interactions with specific phage (and host) proteins. 2. Identify by genetic and biochemical means, known and yet unknown proteins and RNAs that interact with different """"""""domains"""""""" of these origin regions. 3. Establish whether the secondary origin mentioned above and/or additional preferred replication sites identified by other investigation initiate de novo or from recombinational intermediates. 4. Analyse by mutational and subsequent biochemical studies the roles of various (T4 and E. coli) recombination genes and proteins in different steps of recombinational pathways, specifically in those steps that lead to initiation of replication forks. 5. Analyse the roles of these proteins and DNA structure in mutagenesis. 6. Determine the role of membrane proteins and potential membranes attachment of DNA in """"""""origin initiation"""""""" and/or in """"""""recombinational initiation."""""""" 7. Analyse interactions of replication functions and packaging functions. These studies will contribute to understanding of controls operating on DNA transactions at many levels, and on virus-host interactions. Such knowledge should be important in drug treatments of diseases and in attempting to """"""""cure"""""""" genetic diseases with appropriate DNA while avoiding possible mutagenic or carcinogenic effects.
