Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase beta. 2010

Ken Hirano, and Yuichiro Yoshida, and Tomomi Ishido, and Yukihisa Wada, and Naoji Moriya, and Naoshi Yamazaki, and Yoshiyuki Mizushina, and Yoshinobu Baba, and Mitsuru Ishikawa
Nano-Bioanalysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Takamatsu, Kagawa 761-0395, Japan. hirano-ken@aist.go.jp

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase beta (pol beta) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent(R) (exo-), DNA polymerase IIIalpha and the Klenow fragment, and the mammalian polymerases DNA polymerase alpha and human DNA polymerase delta, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol beta. The kinetic parameters K(m) and k(cat) were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol beta. We have demonstrated for the first time that mammalian pol beta can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol beta is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.

UI MeSH Term Description Entries
D009711 Nucleotides The monomeric units from which DNA or RNA polymers are constructed. They consist of a purine or pyrimidine base, a pentose sugar, and a phosphate group. (From King & Stansfield, A Dictionary of Genetics, 4th ed) Nucleotide
D004253 DNA Nucleotidylexotransferase A non-template-directed DNA polymerase normally found in vertebrate thymus and bone marrow. It catalyzes the elongation of oligo- or polydeoxynucleotide chains and is widely used as a tool in the differential diagnosis of acute leukemias in man. EC 2.7.7.31. Terminal Addition Enzyme,Terminal Deoxyribonucleotidyltransferase,Deoxynucleotidyl Transferase,Deoxynucleotidyltransferase,Desoxynucleotidyl Transferase,Desoxynucleotidyltransferase,Tdt Antigen,Terminal Deoxynucleotidyl Transferase,Terminal Deoxyribonucleotidyl Transferase,Addition Enzyme, Terminal,Antigen, Tdt,Deoxynucleotidyl Transferase, Terminal,Deoxyribonucleotidyl Transferase, Terminal,Deoxyribonucleotidyltransferase, Terminal,Enzyme, Terminal Addition,Nucleotidylexotransferase, DNA,Transferase, Deoxynucleotidyl,Transferase, Desoxynucleotidyl,Transferase, Terminal Deoxynucleotidyl,Transferase, Terminal Deoxyribonucleotidyl
D004258 DNA Polymerase III A DNA-dependent DNA polymerase characterized in E. coli and other lower organisms but may be present in higher organisms. Use also for a more complex form of DNA polymerase III designated as DNA polymerase III* or pol III* which is 15 times more active biologically than DNA polymerase I in the synthesis of DNA. This polymerase has both 3'-5' and 5'-3' exonuclease activities, is inhibited by sulfhydryl reagents, and has the same template-primer dependence as pol II. DNA Polymerase delta,DNA-Dependent DNA Polymerase III,DNA Pol III,DNA Dependent DNA Polymerase III,Polymerase III, DNA,Polymerase delta, DNA
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D015342 DNA Probes Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA-DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections. Chromosomal Probes,DNA Hybridization Probe,DNA Probe,Gene Probes, DNA,Conserved Gene Probes,DNA Hybridization Probes,Whole Chromosomal Probes,Whole Genomic DNA Probes,Chromosomal Probes, Whole,DNA Gene Probes,Gene Probes, Conserved,Hybridization Probe, DNA,Hybridization Probes, DNA,Probe, DNA,Probe, DNA Hybridization,Probes, Chromosomal,Probes, Conserved Gene,Probes, DNA,Probes, DNA Gene,Probes, DNA Hybridization,Probes, Whole Chromosomal
D051381 Rats The common name for the genus Rattus. Rattus,Rats, Laboratory,Rats, Norway,Rattus norvegicus,Laboratory Rat,Laboratory Rats,Norway Rat,Norway Rats,Rat,Rat, Laboratory,Rat, Norway,norvegicus, Rattus
D019793 Fluorescein A phthalic indicator dye that appears yellow-green in normal tear film and bright green in a more alkaline medium such as the aqueous humor. Fluorescein Sodium,Sodium Fluorescein,C.I. 45350,Colircusi Fluoresceina,D & C Yellow No. 7,D & C Yellow No. 8,D and C Yellow No. 7,D and C Yellow No. 8,D&C Yellow No. 7,D&C Yellow No. 8,Diofluor,Disodium Fluorescein,Fluor-I-Strip A.T.,Fluorescein Dipotassium Salt,Fluorescein Disodium Salt,Fluorescein Monosodium Salt,Fluorescite,Fluorescéine sodique Faure,Fluorets,Ful-Glo,Funduscein,Minims Fluorescein Sodium,Minims Fluoresceine,Minims Stains,Optifluor Diba,Uranine,Dipotassium Salt, Fluorescein,Disodium Salt, Fluorescein,Fluor I Strip A.T.,Fluorescein Sodium, Minims,Fluorescein, Disodium,Fluorescein, Sodium,Fluoresceina, Colircusi,Fluoresceine, Minims,Ful Glo,Monosodium Salt, Fluorescein,Sodium, Fluorescein
D019951 DNA Polymerase beta A DNA repair enzyme that catalyzes DNA synthesis during base excision DNA repair. DNA Polymerase IV,Polymerase IV, DNA

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