We examined the ribonuclease H (RNase H) specificity of human immunodeficiency virus reverse transcriptase (HIV-RT) using heteropolymeric RNAs hybridized to complementary DNAs. Experiments were performed in the presence of excess challenger polymer (poly(rA)-oligo(dT)) to reveal cleavages resulting from single enzyme binding events. Previous results suggested that initial RNase H directed cleavages were a fixed distance from a DNA primer terminus recessed on an RNA template, i.e. determined by the binding position of the polymerase active site. The influences of recessed RNA termini were not evaluated. In current experiments, RNAs that were 30, 42, or 50 nucleotides long were hybridized to the same 88 nucleotide long complementary DNA, such that the 5' terminal nucleotide of each RNA was hybridized to the 29th nucleotide from the 3' end of the DNA. In all three cases the RNA was initially cleaved between the 19th and 21st nucleotides from its 5' end. Thus, cleavage was not coordinated by the recessed 3' terminus of the RNA. Subsequent cleavages in either direction on the RNA were also observed. An insertion within the RNA that moved the preferred initial cut sequence 10 nucleotides further from the 5' end of the RNA decreased but did not abolish cleavage at the sequence. However, changing the nucleotide sequence in the region of the preferred cleavage either by the insertion experiment or mutagenesis did not significantly alter its capacity for cleavage. These results demonstrated a dominant position preference, plus a sequence priority. In another experiment, a 25 nucleotide long DNA was hybridized such that its 3' terminal nucleotide was 9 nucleotides from the 5' end of a 60 nucleotide complementary RNA. The preferred RNA cleavage sequence discussed above, was 10-14 nucleotides upstream of the 3' end of the DNA. However, initial cleavages occurred 17-20 nucleotides from the DNA 3' end, consistent with cleavage being coordinated by the recessed 3' terminus of the DNA primer.
We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs). Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer. The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H. RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated. Results showed that HIV- and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products. Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity. Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity. The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater. They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand. In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed.
We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 ,M, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.
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