FIGURE 2.

Phi29 DNA polymerase 3′→5′ exoribonuclease activity on the RNA–DNA hybrids. The RNA–DNA hybrid hydrolysis studies were carried out under the conditions described in “Materials and Methods,” using 5′-end-labeled RNA–DNA hybrids. The DNA moiety of the hybrids was a preformed circular single stranded DNA molecule PP1, PP2, or PP3, complementary to the 5′ end (A), center part (B), or 3′ end (C) of the RNA1 molecule. The incubation mixtures, containing various RNA1–PP hybrids and Phi29 DNA polymerase, were incubated at 37°C. Aliquots were removed in timed intervals and analyzed by electrophoresis through denaturing 8% polyacrylamide gel. The labeled RNA1–PP hybrid substrates are shown above the gels. The radioactive label is indicated by the star. “a” represents the hydrolysis products of 5′-end-labeled RNA1-PP hybrids.