The research program deals with mechanisms for regulating the activities of genes. Its broad implications are for understanding gene regulation in developing systems and infection processes, and for precise interventions concerning protein-nucleic acid interactions and gene activity. The specific system under analysis involves the bacteriophage SPO1 encoded chromatin-forming protein TF1, which is a homolog of the ubiquitous prokaryotic type II DNA-binding proteins (DBPII). The DBPII family is remarkable for including proteins that bind to DNA nonspecifically, sequence-specifically and, in the case of TF1, sequence- as well as modified nucleotide-specifically. Experiments dealing with the regulation of phage SPO1 late genes are also proposed. The following lines of work deal with the DNA-binding and -bending protein TF1: (1) We shall determine how the principal contributions to the total affinity of binding are distributed along the large DNA site that binds a single TF1 dimer. (2) The ideal DNA-binding site for TF1 will be defined by a reiterative selection-amplification procedure. (3) TF1 binds preferentially to specific DNA sites in hydroxymethyluracil (hmUra)-containing DNA but not in T-containing DNA. (HmUra substitutes entirely for thymine (T) in phage SPO1 DNA. It occurs in animal and human DNA as a potentially mutagenic oxidation product of T and as an oxidation-plus-deamination product of 5-methylcytosine.) Binding of TF1 to hybrid DNA containing hmUra and T in different combinations is to be analyzed. (4) TF1 will be mapped to its DNA-binding site by site- specific photocrosslinking, using reagents developed in our laboratory. (5) Site-directed mutagenesis experiments seeking to alter TF1 so as to define and manipulate its DNA-binding properties are proposed. (6) TF1 forms higher order DNA complexes, compacting the latter. Experiments to analyze the fine structure of these complexes are proposed. (7) Our work involves a collaboration to determine the 3-dimensional structure of TF1 and TF1-DNA complexes by NMR methods. An in vitro analysis of transcription of SPO1 late genes is proposed. The current focus of interest in these experiments is an analysis of another phage system (T4) in which replication proteins act as transcription factors, utilizing enhancer-like mechanisms with novel properties of protein-protein communication by tracking along DNA. The rationale of a search for the involvement of similar principles in SPO1 late gene regulation is presented and a relevant set of experiments is proposed.
