CHARACTERIZATION OF RNA STRAND DISPLACEMENT SYNTHESIS BY MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASE

The RNase H activity of reverse transcriptase (RT) is presumably requi red to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, s ince RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have inves tigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RN ase H- mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonst rate that the polymerase can displace long regions of RNA in hybrid du plex with DNA but that this activity is approximately 20-fold slower t han DNA displacement and 20-fold slower than non-displacement synthesi s. Furthermore, we find that although certain hybrid sequences seem ne arly refractory to the initiation of RNA displacement, the same sequen ces may not significantly impede synthesis when preceded by a single-s tranded gap. We find that the rate of RNA displacement synthesis by wi ld-type M-MuLV RT is significantly greater than that of the RNase H- R T but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require t he zinc finger domain.