RNASE-H DOMAIN OF MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASERETAINS ACTIVITY BUT REQUIRES THE POLYMERASE DOMAIN FOR SPECIFICITY

The reverse transcriptase-associated RNase H activity of Moloney murin e leukemia virus specifically cleaves within the polypurine tract regi on of the viral genome to generate the primer for plus-strand DNA synt hesis and removes the tRNA primer after minus-strand initiation bg pre ferentially cleaving the RNA one nucleotide before the RNA-DNA junctio n. Moreover, the enzyme is unable to cleave the extended tRNA substrat e at the RNA-DNA junction even at high enzyme concentrations. The RNas e H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extra cts, Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determine d in the plus-strand primer reaction and in the tRNA primer removal re action, Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA -DNA junction. Specificity in both cases could be restored by adding b ack a truncated form of Moloney murine leukemia virus reverse transcri ptase lacking the RNase a domain, These results implicate the polymera se domain as a specificity determinant for the RNase H activity of rev erse transcriptase. The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RN ase H domain alone nor the RNase H domain coupled to the polymerase do main in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation.