NUCLEASE ACTIVITIES OF MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASE - MUTANTS WITH ALTERED SUBSTRATE SPECIFICITIES

RNases H are traditionally thought to degrade RNA only in RNA-DNA hybr id form. We found that the wild-type Moloney murine leukemia virus (M- MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-R NA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel t echniques. Escherichia coli RNase H does not degrade the RNA-RNA duple x in this assay, while E. coli RNase III, a double-strand-specific rib onuclease, does. The apparent specific activity of M-MuLV RT on RNA-RN A duplexes is similar to that on RNA-DNA hybrids. Neither the DNA poly merase domain nor the RNase H domain of RT expressed individually exhi bited this RNA-RNA activity. We have generated a series of mutations i n the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were a s active as the wild type in the oligo(dT):poly(rA) DNA polymerase ass ay, and many retained both nuclease activities. Two enzymes with mutat ions at the carboxyl terminus of the RNase H domain retained RNA-DNA a ctivity, but not RNA-RNA activity. Another mutant enzyme showed the op posite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activit y. Thus, we were able to genetically separate the two activities. Thes e results may be helpful in defining enzyme-substrate interactions.