CLEAVAGE SPECIFICITIES OF MOLONEY MURINE LEUKEMIA-VIRUS RNASE-H IMPLICATED IN THE 2ND STRAND TRANSFER DURING REVERSE TRANSCRIPTION

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integra tion. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define th e maximal 3' and 5' ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reactio n and on model substrates in vitro, RNase H cleaves the genomic RNA te mplate between the second and third ribonucleotides 5' of the U5/PBS j unction, but other minor cleavages between 1 and 10 nucleotides 5' of this junction are also observed. Similar experiments examining the spe cificity of RNase H for tRNA primer removal revealed that cleavage gen erally leaves a ribo A residue at the 5' end of minus strand DNA. Thes e observations suggest that three bases are typically duplicated on th e ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimi cking the structure of this intermediate demonstrate that reverse tran scriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.