We are continuing our study of the E. coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase- helicase, RNase H, and DNA ligase. Assembly of the polymerase and accessory proteins on the primer template. We have previously used cross-linking of proteins to photo-activatable residues at specific nucleotides in the primer to determine the position of polymerase and each of the accessory proteins on the primer. In order to position these proteins on the template strand, and to study the steps in the assembly of the complex by a gel-mobility shift assay, we are conducting a systematic analysis to determine the minimum size primer- template that will allow the proper assembly of the complex. Construction and analysis of mutations in T4 DNA polymerase. T4 DNA polymerase has extensive amino acid sequence similarity with a large family of prokaryotic and eukaryotic DNA polymerases. We are using in vitro mutagenesis to determine the function of these conserved regions and to try to identify regions of the polymerase important for its interaction with the other T4 replication proteins. We have constructed a polymerase lacking the editing exonuclease by changing a conserved amino acid in the N-terminal region. This mutant retains its polymerase activity and interacts normally with the accessory proteins, T4 phage with the exonuclease defective polymerase gene have a very high rate of mutation in vivo. Function of the T4 gene 59 helicase assembly factor. The addition of 59 protein to the other seven replication proteins gives a marked increase in synthesis on duplex DNA. We are currently studying how and where this synthesis is initiated. T4 RNaseH. We have identified a T4 gene encoding an RNaseH, and shown that the purified protein removes RNA primers in vitro. We have constructed a phage mutant with a deletion within the RNaseH gene, and shown that this gene is essential in a host that is lacking both RNaseH and 5 to 3 exonuclease of Pol I. Structure of the T4 replication proteins. We have begun a collaboration with Craig Hyde, NIAMS, to try to crystallize these proteins.
