ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

点击翻译按钮获取中文摘要

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. The structure of T4 RNaseH, a 5' to 3' RNA:DNA and DNA:DNA nuclease, has been solved by heavy atom MIR to 2.8 angstroms for an orthorhombic crystal form. A monoclinic form of higher resolution was then solved by molecular replacement using the refined model. The difference electron density map of the orthorhombic crystal form with BaCl2 replacing MgCl2 clearly shows a single barium binding site in the putative active site of the enzyme, surrounded by the amino acids that are most highly conserved in the prokaryotic and eukaryotic nucleases related to T4 RNaseH. We are constructing single site mutations to test our model and produce an inactive enzyme to crystalize with its duplex substrates. Two orthorhombic crystal forms of the gene 59 helicase assembly protein diffract to high resolution. Function and structure of the T4 primase- helicase complex. We are studying the mechanism by which the gene 59 protein stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins, using affinity chromatography, gel filtration, and chemical cross-linking. ATP increases the cross- linking of both the 41 and 59 proteins to photoactivatable DNA substrates, and cannot be replaced by ATP'S; 59 and 41 proteins each cross-link to the DNA to a much greater extent in the presence of the other protein. Structure and function of the 737-772 region of T4 DNA polymerase. Our structural analysis suggests that the 737-772 region, previously been shown to have effects on polymerase fidelity, may represent a pair of helices bent at G753, and interacting intramolecularly with one another via hydrophobic interactions, or alternatively, could be a dimerization domain between two polymerases. Site-direct mutagenesis is being used to test predictions of these models.