ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

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In the E. coli bacteriophage T4 model system for duplex DNA replication, efficient DNA replication in vitro is achieved with seven purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43 product), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, and the genes 41 and 61 proteins, which together function as a primase and a helicase. Polymerase-Accessory Protein Interactions. The three accessory proteins function as a sliding clamp to keep the polymerase bound to the primer-template. Polymerase and the 44/62 protein bind independently to oligonucleotide-primed ssDNA in the absence of ATP. Subsequent binding of 45 protein requires ATP as well as both polymerase and the 44/62 protein complex. Strand Displacement Synthesis on Forked Templates. A 50 base region on the side of a synthetic forked DNA substrate corresponding to the lagging strand template allows synchronous and rapid interaction of leading strand displacement synthesis, indicating that the leading strand polymerase and/or its accessory proteins is in contact with both template strands at the replication fork. The preformed fork also permits 41 protein- dependent strand displacement synthesis on some molecules in the absence of 32 protein. These results are consistent with a model in which 41 protein helicase controls unwinding of the duplex, while 32 protein acts to prevent reannealing of the strands unwound. Primase-Helicase Interactions with Other Replication Proteins. A tryptic fragment of 41 protein, missing 17 or 20 amino acids from the C-terminus, retains the primase and helicase activities of the intact enzyme, but is unable to catalyze these activities in the presence of the other replication proteins. Primer synthesis by 61 protein, and either intact 41 protein or the tryptic fragment, is inhibited by high concentrations of 32 protein. The polymerase accessory proteins reverse this inhibition with intact 41 protein but not with the 41 fragment. Thus, alteration of the C-terminus of 41 protein prevents an interaction with the accessory proteins which is necessary for primer synthesis on 32 protein coated DNA.