The first step in conversion of phiX174 single-stranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate. It is now demonstrated that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a phiX174 circle. The single dnaB protein molecule present on each DNA circle participated in initiation of each of the RNA primers, which appears to be aligned at regular intervals along the template strand. It is proposed that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a 'mobile promotor' signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the E. coli chromosome and in the initiation of primers for nascent (Okazaki) fragments at a replication fork.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 1977|
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