title: Construction of single amino acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'-->3' exonuclease activity.
abstract: Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.
authors.affiliation: Gen-Probe Incorporated, San Diego, CA 92121, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Riggs
authors.forename: S
authors.initials: S
authors.lastname: Tudor
authors.forename: M
authors.initials: M
authors.lastname: Sivaram
authors.forename: S H
authors.initials: SH
authors.lastname: McDonough
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics* / Q000302:isolation & purification; D004274:DNA, Recombinant; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000235:genetics
chemical_list: D000596:Amino Acids; D004274:DNA, Recombinant; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D013381:Subtilisins