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 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 priming proteins. 61 protein alone has a limited primase activity and 41 protein alone has a DNA unwinding (helicase) activity. The two proteins together form a functional complex that catalyzes both activities much more efficiently than the individual proteins. Although 61 protein alone synthesizes mainly the dimers pppAC and pppGC, only the rare oligonucleotides which begin with G and are long enough to hybridize stably to the DNA template are able to initiate new DNA chains in the absence of 41 protein, in contrast to the pentanucleotide primers found at the ends of DNA with the 41 and 61 proteins together. On T4 DNA, which has hydroxymethyl cytosine in place of cytosine, DNA chain initiation requires both the 41 and 61 proteins. We have proposed and are currently testing a model in which the interaction of the polymerase-accessory protein complex with the primase-helicase controls the selection of priming sites for the initiation of new fragments on the lagging strand. We have constructed plasmids expressing 41 protein with N-terminal and C-terminal deletions and have purified these proteins to identify 41 protein domains required for its primase, helicase, and DNA-dependent nucleotidase activities. We are studying the factors and sequences regulating the expression of the T4 DNA replication proteins, including genes uvsX (recombination protein) through genes 41, 61, and dam (DNA adenine methylase). We have located two strong transcription promoters 700-800 bp upstream of uvsX which are active in vivo and in vitro. A transcription terminator between genes uvsX and 41 contributes to the differential levels of these two proteins. The T4 dam protein methylates the A in the sequence GATC. Expression of the T4 dam gene in pBR322-based plasmids causes E. coli cells to grow poorly, particularly if they lack RNase H.