title: Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase beta.
abstract: 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.
authors.affiliation: 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
authors.forename: Ken
authors.initials: K
authors.lastname: Hirano
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Yoshida
authors.forename: Tomomi
authors.initials: T
authors.lastname: Ishido
authors.forename: Yukihisa
authors.initials: Y
authors.lastname: Wada
authors.forename: Naoji
authors.initials: N
authors.lastname: Moriya
authors.forename: Naoshi
authors.initials: N
authors.lastname: Yamazaki
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Mizushina
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ishikawa
mesh_terms: D000818:Animals; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D051381:Rats
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D009711:Nucleotides; D004253:DNA Nucleotidylexotransferase; D004258:DNA Polymerase III; D019951:DNA Polymerase beta; D019793:Fluorescein