ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

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We are studying the E. coli bacteriophage T4 as a model system for duplex DNA replication. Efficient DNA synthesis in vitro can be achieved with a mixture of 7 purified proteins encoded by T4 phage, which include T4 DNA polymerase (gene 43 product), the gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, and the gene 41 and gene 61 priming proteins. We have cloned the genes for the T4 61 and 41 primase proteins and designed new procedures for the large-scale purification of each protein. At very high concentrations, 61 protein alone catalyzes some oligonucleotide synthesis on single-stranded DNA templates, yielding mainly dinucleotides (pppAC) in contrast to the pentanucleotides formed by the combined action of the 61 and 41 primase proteins. Oligonucleotides made without 41 protein prime DNA synthesis by T4 polymerase and its accessory proteins, but both primer and DNA synthesis are greatly stimulated by the 41 protein. The DNA unwinding activity (helicase) of 41 protein is increased more than 30-fold by 61 protein, which has no independent unwinding activity. We are screening mutant forms of 41 protein, produced by existing mutants and by in vitro mutagenesis of the cloned gene, to identify regions of 41 protein required for its primase, helicase, and DNA-dependent nucleotidase activities. Plasmids encoding the T4 uvsX recombination protein decrease the UV sensitivity of T4 mutants in the uvsX gene. Purified uvsX protein catalyzes ATP hydrolysis to ADP and AMP and strand exchange between homologous single-stranded circular and linear duplex DNA. Strand exchange is stimulated by T4 gene 32 protein and by a 3' single-stranded extension on the duplex DNA. We are using the plasmids containing the T4 uvsX and 41 helicase genes and those containing the 61 primase and DNA adenine methylase genes to identify sequences and factors regulating the expression of the T4 DNA in these two neighboring regions.