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 transcripta se (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-D NA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the l ast 19 amino acids, were expressed. In both cases, these RTs made mult iple cuts in PPT-containing substrates, whereas wild-type cleavages oc curred primarily at sites consistent with the distance between the pol ymerase and RNase H active sites. Primer extension assays performed wi th the chimeric RTs, an RNase H-minus RT, and wild-type showed that th e presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wildtype RT was bound to primer-template, two reta rded bands could be detected in band-shift assays. In the absence of p rimer 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 supershifted complex. Thi s suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but th e chimeric RTs were somewhat less defective than the RNase H-minus mut ant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer -template.