Formation of rolling-circle molecules during phi X174 complementary strand DNA replication.
The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n"). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.
D010584:Bacteriophage phi X 174 / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D007700:Kinetics
D004279:DNA, Viral; D004247:DNA; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D004265:DNA Helicases