title: RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases.
abstract: DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.
authors.affiliation: Third Wave Technologies, Inc., Madison, Wisconsin 53719, USA. wma@twt.com
authors.forename: W P
authors.initials: WP
authors.lastname: Ma
authors.forename: M W
authors.initials: MW
authors.lastname: Kaiser
authors.forename: N
authors.initials: N
authors.lastname: Lyamicheva
authors.forename: J J
authors.initials: JJ
authors.lastname: Schaefer
authors.forename: H T
authors.initials: HT
authors.lastname: Allawi
authors.forename: T
authors.initials: T
authors.lastname: Takova
authors.forename: B P
authors.initials: BP
authors.lastname: Neri
authors.forename: V I
authors.initials: VI
authors.lastname: Lyamichev
mesh_terms: D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D015850:Interleukin-6 / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D015850:Interleukin-6; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012313:RNA; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases