title: Chimeric thermostable DNA polymerases with reverse transcriptase and attenuated 3'-5' exonuclease activity.
abstract: The synthesis of accurate, full-length cDNA from low-abundance RNA and the subsequent PCR amplification under conditions which provide amplicon that contains minimal mutations remain a difficult molecular biological process. Many of the challenges associated with performing sensitive, long RT/PCR have been alleviated by using a mixture of DNA polymerases. These mixtures have typically contained a DNA polymerase devoid of 3'-5' exonuclease, or "proofreading", activity blended with a small amount of an Archaea DNA polymerase possessing 3'-5' exonuclease activity, since reverse transcriptases lack 3'-5' exonuclease activity and generally have low fidelity. To create a DNA polymerase with efficient reverse transcriptase and 3'-5' exonuclease activity, a family of mutant DNA polymerases with a range of attenuated 3'-5' exonuclease activities was constructed from a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These "designer" DNA polymerases were fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues resulted in proteins in which DNA polymerase activity was unaffected, while proofreading activity ranged from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicated that the mutations affected catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5-15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR. The utility of these enzymes was evaluated in a RT/PCR assay to generate a 1.7 kb amplicon from HIV-1 RNA.
authors.affiliation: Program in Core Research, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501, USA. nancy.schoenbrunner@roche.com
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Schönbrunner
authors.forename: Ellen H
authors.initials: EH
authors.lastname: Fiss
authors.forename: Olga
authors.initials: O
authors.lastname: Budker
authors.forename: Susanne
authors.initials: S
authors.lastname: Stoffel
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Sigua
authors.forename: David H
authors.initials: DH
authors.lastname: Gelfand
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Myers
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013698:Templates, Genetic; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000201:enzymology; D013824:Thermus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases