Complexes of Rep protein with ATP and DNA as a basis for helicase action.
The rep protein of Escherichia coli, a helicase that unwinds duplex DNA at a replication fork (Kornberg, A., Scott, J. F., and Bertsch, L. L. (1978) J. Biol. Chem. 253, 3298-3304), forms binary complexes with ATP and with DNA, and ternary complexes with both. ATP (or dATP) is bound at a single site with a dissociation constant (KD) near 10(-7) M. Other ribonucleoside triphosphates and deoxyribonucleoside triphosphates compete for the same site with far lower affinities. The protein forms a binary complex with single-stranded DNA and with duplex DNA, each at distinctive sites. Binding to single-stranded DNA covers a stretch of approximately 20 nucleotides, destabilizes secondary structure, and facilitates reannealing of complementary single strands. Ternary complexes of rep protein with ATP and DNA are manifested by ATP hydrolysis and by binding of labeled components. Nonhydrolyzed ATP analogs are useful aids for isolation and studies of such complexes. Unlike rep protein's processive action as a helicase at a replication fork, its action on single-stranded DNA is distributive, with ATP hydrolysis accelerating dissociation of the protein from the complex. These and related studies serve as guides to understanding the multiple interactions of rep protein with its ATP and DNA ligands that enable it to unwind duplex DNA at a replication fork.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate; D010584:Bacteriophage phi X 174 / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000494:pharmacology; D004338:Drug Combinations; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D011089:Polydeoxyribonucleotides; D011485:Protein Binding; D012265:Ribonucleotides / Q000494:pharmacology; D013379:Substrate Specificity
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D004338:Drug Combinations; D029968:Escherichia coli Proteins; D011089:Polydeoxyribonucleotides; D012265:Ribonucleotides; C506744:rep protein, E coli; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases