RNASE H ACTIVITY OF REVERSE TRANSCRIPTASES ON SUBSTRATES DERIVED FROMTHE 5' END OF RETROVIRAL GENOME

RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were use d to study the specificity of the RNase H activities of HIV, AMV (avia n myeloblastosis virus), and MMuLV reverse transcriptases. These subst rates were selected because they represent the site for the first temp late switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequenc e of the RNA/DNA substrate. The minimal size of hybrid recognized by t he RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover , the size of the residual 5'-undigested RNA after completion of the R Nase H reaction depended on the position of the DNA annealed to the ge nomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained follow ing digestion by HIV reverse transcriptase, and 21-24 nucleotides foll owing digestion by AMV reverse transcriptase and MMuLV reverse transcr iptase. On the other hand, with all three enzymes, smaller sized hybri ds remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of R Nase H to efficiently digest at the 5' end of hybrid structures. Surpr isingly, hybridization to the RNA template, of a DNA oligomer that ext ended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of acti on of RNase H at the 5' end of the genomic RNA should be taken in cons ideration in studies of the first template switch.