Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA.
Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D010584:Bacteriophage phi X 174 / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis*; D004338:Drug Combinations; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004338:Drug Combinations; D029968:Escherichia coli Proteins; D014764:Viral Proteins; C506744:rep protein, E coli; D004258:DNA Polymerase III; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases