Fluorescence recovery assay: a continuous assay for processive DNA polymerases applied specifically to DNA polymerase III holoenzyme.
A continuous assay was developed for processive DNA polymerases. The specific enzyme used to develop the assay was the most processive polymerase known, Escherichia coli DNA polymerase III holoenzyme. The assay was based upon the recovery of the intrinsic fluorescence of single-stranded DNA binding protein (SSB) as it was displaced from the DNA template during DNA synthesis. The intrinsic fluorescence of SSB was quenched by as much as 80% when it bound to single-stranded DNA. As the DNA was replicated, SSB was displaced and recovered its fluorescence. The amount of fluorescence recovered was directly proportional to the amount of DNA synthesized and was used to quantitate the rate of DNA synthesis. However, since 50 to 60 nucleotides must be replicated for every SSB tetramer released, the assay is expected to work best for processive DNA polymerases. The only requirement for using this assay with other DNA polymerases is that they be able to synthesize DNA on a template coated with SSB. The replication SSBs do not pose an obstacle to the assay because they all appear to have intrinsic fluorescence that is sensitive to their ssDNA-bound state.
Griep
M A
MA
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA. mgriep@unl.edu
D001426:Bacterial Proteins / Q000032:analysis*; D004258:DNA Polymerase III / Q000032:analysis*; D004261:DNA Replication / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000494:pharmacology; D007700:Kinetics; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013698:Templates, Genetic
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005971:Glutamates; D011189:Potassium Chloride; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase