title: Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution.
abstract: DNA polymerases recognize their substrates with exceptionally high specificity, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
authors.forename: Farid J
authors.initials: FJ
authors.lastname: Ghadessy
authors.forename: Nicola
authors.initials: N
authors.lastname: Ramsay
authors.forename: François
authors.initials: F
authors.lastname: Boudsocq
authors.forename: David
authors.initials: D
authors.lastname: Loakes
authors.forename: Anthony
authors.initials: A
authors.lastname: Brown
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vaisman
authors.forename: Roger
authors.initials: R
authors.lastname: Woodgate
authors.forename: Philipp
authors.initials: P
authors.lastname: Holliger
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003855:Deoxyribose / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D007700:Kinetics; D046228:Microarray Analysis; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011740:Pyrimidine Dimers / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism
chemical_list: D015342:DNA Probes; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D003856:Deoxyuracil Nucleotides; D007211:Indoles; D011740:Pyrimidine Dimers; D012235:Rhodamines; D013873:Thionucleotides; C065361:2'-deoxy-7-deazaguanosine triphosphate; C027078:deoxyuridine triphosphate; D003855:Deoxyribose; D001710:Biotin; D004247:DNA; D019914:Taq Polymerase; D016650:Fluorescein-5-isothiocyanate; C026600:2'-deoxyadenosine triphosphate; C090067:5-nitroindole