THE USE OF DNA AND RNA OLIGONUCLEOTIDES IN HYBRID STRUCTURES WITH LONGER POLYNUCLEOTIDE CHAINS TO PROBE THE STRUCTURAL REQUIREMENTS FOR MOLONEY MURINE LEUKEMIA-VIRUS PLUS STRAND PRIMING

Plus strand priming during retroviral reverse transcription requires s pecific cleavage within the polypurine tract of the viral genome by th e reverse transcriptase-associated RNase H. Previously it has been sho wn that a 190-base RNA-DNA hybrid containing the Moloney murine leukem ia virus polypurine tract can serve as a substrate for the priming rea ction. To investigate the structural requirements for the reaction, a series of DNA oligonucleotides was hybridized to the 190-base single s tranded RNA and tested as substrates for RNase H. At low enzyme concen trations, the sites of cleavage are located 17-23 nucleotides from the 3'-end of the DNA oligonucleotide, consistent with the observations o f others that binding of the DNA polymerase at a primer terminus fixes the position of cleavage by RNase H. At higher enzyme concentrations, additional cleavages are observed in the RNA 3' of these sites, but t here is no preference for cleavage at the plus strand origin. In contr ast to the results with DNA oligonucleotides, hybridization of RNA oli gonucleotides containing the polypurine tract to the 190-base single s tranded DNA generates substrates that are cleaved at the origin and ef ficiently extended into DNA. An RNA oligonucleotide hybridized downstr eam of the polypurine tract is cleaved but not extended. These results support the view that RNase H cleavage to generate the plus strand pr imer is uncoupled from minus strand DNA synthesis.