FIGURE 4.

Exoribonuclease activity with DNA-RNA chimeric substrates; comparison of wild-type and mutant proteins. Activity assays were performed using two 15-mer chimeric substrates as described under “Experimental Procedures.” A, activity assays with Chi 1 (5′-dTdTdTdTdTdTdTdTdTdTdTrCrCdTdT-3′) were the 3rd and 4th positions from the 3′-end are occupied by ribonucleotides (rC), and the other 13 are deoxyribonucleotides (dT). B, activity assays with Chi2 (5′-dTdTdTdTdTdTdTdTdTdTdTrCdTrCdT-3′) were the 2nd and 4th positions from the 3′-end are occupied by ribonucleotides, and the other 13 are deoxyribonucleotides. The mutants used and their respective protein concentrations are shown. The wild-type enzyme was used as control. Samples were taken during the reaction at the time points indicated, and reaction products were analyzed in a 20% polyacrylamide, 7 m urea gel. Control reactions with no enzyme added (Ctrl) were incubated at the maximum reaction time for each protein. Length of substrates and degradation products are indicated in the figure.