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.
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 complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.
Guo
J
J
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wu
W
W
Yuan
Z Y
ZY
Post
K
K
Crouch
R J
RJ
Levin
J G
JG
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H