Defects in primer-template binding, processive DNA synthesis, and RNase H activity associated with chimeric reverse transcriptases having the murine leukemia virus polymerase domain joined to Escherichia coli RNase H

Biochemistry
J GuoJ G Levin

Abstract

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this ...Continue Reading

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Citations

Nov 25, 2005·The Journal of Biological Chemistry·Sharon J SchultzJames J Champoux
Jul 20, 2007·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Aravind Basavapathruni, Karen S Anderson
Aug 25, 2000·The Journal of Biological Chemistry·M WisniewskiR A Bambara
Apr 25, 1997·The Journal of Biological Chemistry·C PalaniappanR A Bambara
Nov 27, 1999·The Journal of Biological Chemistry·S J SchultzJ J Champoux
May 23, 2002·The Journal of Biological Chemistry·Michele WisniewskiRobert A Bambara
Oct 19, 2007·Nucleic Acids Research·Yasumasa IwataniJudith G Levin
Oct 10, 1998·Journal of Virology·S J Steele, H L Levin

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