Experiments are proposed to ask questions about how virus chromosomes are selected and packaged during assembly. The system used is bacteriophage lambda, for which powerful genetic and biochemical methods of analysis are available. One aspect of chromosome recognition that is poorly understood is the basis for the recognition of the cohesive end site (cos) of the DNA by terminase which generates the cos-terminase complex called complex I. The proposed experiments will define the terminase domain involved in cleavage of the cutting site, cosN. cosN point mutations will give detailed information about the role of specific cosN base pairs in recognition by terminase. The initial interaction of terminase with the binding site, cosB, will be studied and the role of the host factor elucidated. There is a segment of DNA between cosN and cosB that is of unknown significance. Experiments will test whether this segment is involved in specific interactions with packaging proteins or whether it is simply a spacer. The protein(s) involved in interaction with the segment will be identified. The role of the host factor IHF in 21 terminase action will be examined. IHF-dependent mutants (her) of 21 will be examined for host factor requirements. Mutants defective in the Lambda host factor (HF) will be sought and studied. Interactions involved in the binding of the prohead by complex II will also be examined. One important goal is to identify the prohead protein that binds to terminase during complex II formation. The role of the accessory protein gpFI will be studied to understand how it alters the intracellular environment so that packaging can occur. The interaction of gpFI with the phage-coded ben endonuclease, and with host factor(s) will be studied. The proposed work will be of general significance for virus biology, including pathogenic viruses. Fundamental information on DNA-protein interactions, assembly mechanisms and virus-host interactions will result.
