title: Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase.
abstract: Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.
authors.affiliation: Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
authors.forename: M J
authors.initials: MJ
authors.lastname: Longley
authors.forename: S E
authors.initials: SE
authors.lastname: Bennett
authors.forename: D W
authors.initials: DW
authors.lastname: Mosbaugh
mesh_terms: D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D004269:DNA, Bacterial / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology*
chemical_list: D004269:DNA, Bacterial; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases