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. Mutations in T4 DNA polymerase. Our studies of an antimutator mutant in T4 DNA polymerase (A737V) indicate that the accuracy of the polymerase can be altered by changing the rate at which the growing strand of the duplex moves between the polymerase and exonuclease active sites on the enzyme. This single amino acid substitution decreases the processivity of the polymerase activity and increases the processivity of the proofreading exonuclease activity. These processivity changes are reversed by the compensating L771F mutation. Interactions between replication proteins. We are studying how interactions between proteins in the replication complex regulate synthesis on the leading and lagging strands. DNA templates with photo-activatable cross-linking residues in the fork ahead of the primer have been constructed, and are being used to determine whether any of the accessory proteins are in front of polymerase, and to learn how and where the 61, 41, and 59 protein primase-helicase components assemble at the fork. In studies of the mechanism by which the gene 59 protein stimulates the 41/61 protein primase-helicase, we have demonstrated a physical interaction between the 59 and 41 proteins in the absence of DNA. T4 RNaseH. In a nonpermissive (RNaseH defective) host, a T4 mutant with a deletion in the phage RNaseH gene makes few viable progeny, and accumulates short DNA chains characteristic of unligated lagging strand fragments. We are using purified T4 RNaseH to study how primer removal and gap filling are coordinated. Structure of the T4 replication proteins. We are collaborating with Craig Hyde, NIAMS, to try to crystallize these proteins.
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