Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein.
The Escherichia coli primosome is a mobile multiprotein DNA replication-priming apparatus that assembles at a specific site (termed a primosome assembly site (PAS] on single-stranded DNA-binding protein-coated single-stranded DNA. The PRI A protein (factor Y, protein n') is a PAS sequence-specific (d)ATPase as well as a DNA helicase and is believed to direct the assembly of the primosome at a PAS. In this report, the PRI A DNA helicase reaction is dissected in vitro, by use of a strand displacement assay, into three steps with distinct ATP requirements. First, the PRI A protein gains entry to the DNA via an ATP-independent, PAS sequence-specific binding event. Second, the PRI A protein translocates along the single-stranded DNA in the 3'----5' direction at a maximal rate of 90 nucleotides/s. DNA translocation requires ATP hydrolysis. The ATP concentration required to support half of the maximal translocation rate is 100 microM, which is identical to the Km for ATP of the PRI A protein DNA-dependent ATPase activity. Finally, the PRI A protein unwinds duplex DNA. The ATP concentration required for duplex DNA unwinding depends upon the length of the duplex region to be unwound. Displacement of a 24-nucleotide long oligomer required no more ATP than that required for the translocation of PRI A protein along single-stranded DNA, whereas displacement of a 390-nucleotide long DNA fragment required a 10-fold higher concentration of ATP than that required for oligomer displacement.
Lee
M S
MS
Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Marians
K J
KJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011485:Protein Binding
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases