ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

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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. Structure of the T4 DNA replication proteins. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of each of the T4 DNA replication proteins using single crystal X-ray diffraction. We have obtained high resolution data (2.5 angstroms) on large single crystals of RNaseH and the gene 59 helicase assembly protein, and are screening heavy atom derivatives for phasing potential. Assembly of replication proteins on a forked DNA template. We have constructed substrates with a photoactivatable residue in the fork ahead of a primer to learn how and where the polymerase and polymerase accessory proteins and the gene 61, 41, and 59 protein primase-helicase components assemble at the fork. ATP hydrolysis by the 44/62 complex is required to assemble the 45 protein on a primer in a form in which it can move forward on the single-stranded DNA, as shown by its cross-linking to the fork ahead. Polymerase adds to this complex, occupying a position ahead of the 45 protein, and is cross-linked only when there is no gap between the primer and the fork. Function and structure of the T4 primase-helicase complex. We are studying the mechanism by which the gene 59 proteins stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins. We are probing the physical interactions between these proteins by affinity chromatography, centrifugation, and chemical cross- linking. T4 RNaseH. We are using purified T4 RNaseH to determine how primer removal by RNaseH and gap filling by polymerase are coordinated on the lagging strand. During 5' to 3' hydrolysis T4 RNaseH removes oligonucleotides of 1 to 5b. The rate of digestion and length of the products are increased by 32 protein. T4 DNA polymerase synthesis from an upstream fragment stimulates RNaseH hydrolysis on the adjacent fragment.