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J. Biol. Chem., Vol. 255, Issue 9, 4290-4293, May, 1980
NK Sinha, CF Morris and BM Alberts
A wide variety of double-stranded DNA templates are replicated extensively
in an in vitro DNA replication system containing the purified proteins
specified by seven T4 bacteriophage DNA replication genes (32, 41, 43, 44,
62, 45, and 61). In favorable conditions, this multiprotein system
catalyzes the synthesis of several copies of the input DNA template in a
30- to 60-min incubation. The replication forks produced in vitro move in a
highly processive fashion, at approximately the in vivo rate of 500
nucleotides per s. The DNA synthesized on the lagging side of the in vitro
replication fork is made discontinuously, as it is in vivo, giving rise to
"Okazaki pieces" averaging some 10,000 nucleotides in length; in contrast,
DNA is polymerized in a continuous manner on the leading side of the in
vitro fork. Although the mechanism by which the seven-protein in vitro DNA
replication system propagates replication forks closely resembles the in
vivo mechanism, it lacks the capacity to remove RNA primers, to reseal
Okazaki pieces, and to initiate replication forks at defined DNA origins;
supplementation of the system with additional T4-specific replication
proteins will be required to facilitate these latter three functions.
Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system
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