PROPERTIES OF THE 5'-]3' EXONUCLEASE RIBONUCLEASE-H ACTIVITY OF THERMUS-THERMOPHILUS DNA-POLYMERASE

The recombinant 94 kDa Thermus thermophilus DNA polymerase (rTth pol) was found to release [P-33]UMP when incubated with a RNA . DNA hybrid containing a [P-33]UMP-labeled RNA strand. The RNase H activity was optimally active in the presence of low monovalent salt concentrations and when Mn2+ was used as the divalent cation activator. RNase H activity also was observed when Mg2+ replaced the Mn2+, but to a much lesser extent. A 60 nucleotide long, 5'- or 3'-radiolabeled RNA or DNA oligomer hybridized to a complementary DNA oligomer was used to determine the mode of digestion. The radiolabeled RNA . DNA hybrid or DNA . DNA duplex was incubated with rTth pol using various metal ion conditions and different incubation times. The DNA . DNA duplex showed very little enzymatic cleavage by rTth pol regardless of the Mn2+ or Mg2+ concentration. However, nearly complete digestion of the RNA . DNA hybrid was observed over a wide Mn2+ concentration range, thus demonstrating a preferential degradation of the RNA . DNA hybrid vs the DNA . DNA duplex. Time course reactions of the enzymatic digestion of the 3'-labeled RNA . DNA hybrid or DNA . DNA duplex by rTth pol indicated that digestion of the substrates occurred exonucleolytically in the 5'-->3' direction.