Abstract
Plus strand priming during retroviral reverse transcription requires specific cleavage within the polypurine tract of the viral genome by the reverse transcriptase-associated RNase H. Previously it has been shown that a 190-base RNA-DNA hybrid containing the Moloney murine leukemia virus polypurine tract can serve as a substrate for the priming reaction. To investigate the structural requirements for the reaction, a series of DNA oligonucleotides was hybridized to the 190-base single-stranded RNA and tested as substrates for RNase H. At low enzyme concentrations, the sites of cleavage are located 17-23 nucleotides from the 3'-end of the DNA oligonucleotide, consistent with the observations of 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 there is no preference for cleavage at the plus strand origin. In contrast to the results with DNA oligonucleotides, hybridization of RNA oligonucleotides containing the polypurine tract to the 190-base single-stranded DNA generates substrates that are cleaved at the origin and efficiently extended into DNA. An RNA oligonucleotide hybridized downstream of the polypurine tract is cleaved but not extended. These results support the view that RNase H cleavage to generate the plus strand primer is uncoupled from minus strand DNA synthesis.
Highlights
Reverse transcriptases of all retroviruses possess two disshown that a 190-baseRNA-DNA hybrid containing the tinct enzymatic activities, an RNA- or DNA-dependent DNA
Moloney murine leukemia virus polypurine tract can polymerase and a ribonuclease H (RNase H) which degrades serve as a substrate for the priming reactioTno.inves- RNA that is in a hybrid duplexwith DNA
Both activities have tigate the structural requirements for the reaction, a been widely studied yet questions remainconcerning the coorseries of DNA oligonucleotides was hybridized to the dination of the two activities during several steps of reverse
Summary
Indicated) and 3 units of T4 DNA polymerase at 37 "C for 30 min. DNA-RNA hybrids were formed by heating to 65 "C for 3 min in the Materials-M-MuLV reverse transcriptase, RNase H- RTE Polymerase, and DNA sequencing reagents were purchased from United Primer Extension Assays-6-pl portioofntshe enzyme reactions were. RNA oligonucleotides were synthesized tions, T4 polymerase buffer(above) plus 0.33mM dNTPs and units of on a n Applied Biosystems 394DNA/RNA synthesizer using phosphora- enzyme were substituted. Both DNA and RNA oligonucleotides were gel-purified Alkaline Hydrolysis-EDTA was added t o the reactions to a final by electrophoresisthrough 20% polyacrylamide, 8 M ureagels.The concentration of 15mM, and the alkaline hydrolysis carried out inM 0.3 bands wereexcised and theoligonucleotides eluted with 50mM ammo- NaOH at 65 "C for 30 min. 20-1-11reactions were incubatedat 37 "C for 1h analyses with the loRngNA are designatedas follows:M(A,B),where M and stopped by addition of 98% formamide, 10 mM EDTA, with xylene indicates the minus straonfdthe M-MuLV sequence, Bis the coordinate cyanol and bromphenol blue as markers. PB190(-) was linearizedby digestion with PuuII and usaesda template for T7 RNA polymerase to preparae 485 base plusRNA containing the
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