Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase.
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.
Longley
M J
MJ
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Bennett
S E
SE
Mosbaugh
D W
DW
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*
D004269:DNA, Bacterial; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases