title: A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.
abstract: The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003090:Coliphages / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis*; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D008274:Magnesium