REVERSE TRANSCRIPTION OF THE MAURICEVILLE PLASMID OF NEUROSPORA - LACK OF RIBONUCLEASE-H ACTIVITY ASSOCIATED WITH THE REVERSE-TRANSCRIPTASEAND POSSIBLE USE OF MITOCHONDRIAL RIBONUCLEASE-H

The Mauriceville mitochondrial plasmid of Neurospora encodes a reverse transcriptase that synthesizes a full-length cDNA copy of the major p lasmid transcript beginning directly opposite the 3' end of the templa te RNA (Kuiper, M. T. R., and Lambowitz, A. M. (1988) Cell 55, 693-704 ). Here, we show that the Mauriceville plasmid reverse transcriptase h as no detectable RNase H activity and that cDNAs synthesized either by the column-purified reverse transcriptase or by the endogenous revers e transcriptase in purified ribonucleoprotein particles remain in a fu ll-length duplex with the template RNA. The column-purified Mauricevil le plasmid reverse transcriptase initiates cDNA synthesis by using sho rt DNA primers, which remain attached to the 5' end of the (-) strand DNA (Wang, H., Kennell, J. C., Kuiper, M. T. R., Sabourin, J. R., Sald anha, R., and Lambowitz, A. M. (1992) Mol. Cell. Biol. 12, 5131-5144). We rind that these primer DNAs can be precisely removed by S1 nucleas e digestion of the initial cDNA.RNA duplex, suggesting a mechanism whe reby this structure may contribute to primer removal in vivo. Finally, we show that Neurospora mitochondria contain an endogenous RNase H ac tivity, which is present in mitochondrial ribonucleoprotein particle p reparations prior to their purification. This mitochondrial RNase H ca n degrade the endogenous plasmid transcript in ribonucleoprotein parti cles in vitro and could play a similar role in vivo. The finding that the Mauriceville plasmid reverse transcriptase, which is believed to b e a primitive enzyme, has no detectable RNase H activity is consistent with the hypothesis that retroviral reverse transcriptases acquired t heir RNase H domains from a gene encoding a cellular RNase H.