The long-term goal of my laboratory is to understand the relationship between the architecture and function of proteins that participate in DNA replication. We have focused our studies on the T4 DNA replication system because of the extensive genetic, and biochemical and biophysical information that has already been obtained, the fact that this process can be readily studied in vitro, and the belief that it provides a reasonable model for DNA replication in higher organisms. Knowledge of the structure of the proteins involved in T4 DNA replication as well as the organization and regulation of the relevant genes is required before a detailed understanding of functionally significant protein-protein and protein-DNA interactions can be achieved. Accordingly we have determined the nucleotide sequence of several of the structural genes coding for the T4 DNA replication proteins and have identified the control regions that regulate their expression. We have cloned the genes for T4 DNA polymerase as well as accessory proteins in high-level expression vectors and have obtained sufficient amounts of the proteins to carry out structure-function studies. These include crystallization, selective in vitro mutagenesis, limited proteolysis, and physico-chemical studies that should illuminate the nature of their interactions with each other and with DNA. Studies on the T4 single strand DNA binding protein (32P), and essential component of the replication complex, and studies on other single-strand DNA binding proteins are continuing with emphasis on crystallization, 1H- NMR and physical-chemical studies that should show which residues are involved in binding DNA and which regions are necessary for interaction with other proteins in replication complexes. We plan to extend these approaches to eukaryotic systems with emphasis on the 72kd adenovirus- specified DNA binding protein as an example of a eukaryotic single-strand DNA binding protein that may have analogous structure-function relationships as found in the T4 gene 32 protein.
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