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 replicatoin. 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 43 polymerase accessory proteins, and the genes 41 and 61 priming proteins. Primase-Helicase. The 61 and 41 proteins function as a complex with primase and DNA-unwinding (helicase) activities. The proteins act as a helicase to open the duplex ahead of the nascent leading strand, and stop periodically to make the pentanocleotide primers needed to initiate new chains on the lagging strand. We have shown that the 41-61 helicase interacts with a region of greater than 50 nucleotides on the lagging strand template and 40 nucleotides on the leading strand template. The rate of unwinding by the 41/61 helicase is greatest with all four rNTP needed for primer synthesis. The 61 and 41 proteins together make mainly the pentamer primers which initiate new T4 DNA strands in vivo. 61 protein alone has a weak primase activity making predominantly dimers and traces of longer products. In the absence of 41 protein, the gene 32 ssDNA binding prtein strongly stimulates the synthesis of very long RNA (n more than 300) by high concentrations of 61 proteins. Our studies suggest that 41 protein specifically stimulates the synthesis of pentamers beginning with A, prevents the 61 and 32 proteins from elongating the pentamer, but facilitates their elongation into DNA by T4 DNA polymerase and its accessory proteins. Using a gel-DNA retardation assay, we have shown that 41-protein binds tightly to ssDNA only in the presence of bith 61 protein and an NTP. Binding is greatest with ATP or GTP, which serve as cofactors for the 41 protein helicase. RNase H. We have shown that RNase H activity increases 10-fold after T4 infection and have purified this activity from T4 infected cells. This RNase H efficiently removes the pentamer primers synthesized by the 41 and 61 proteins.