STRAND DISPLACEMENT SYNTHESIS CAPABILITY OF MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASE

The accepted model of retroviral reverse transcription includes a circ ular DNA intermediate which requires strand displacement synthesis for linearization and creation of an integration-competent, long terminal repeat-banked DNA product. We have used an in vitro model of this las t step of reverse transcription to examine the role of the viral enzym e, reverse transcriptase (RT), in displacement synthesis. We show that Moloney murine leukemia virus RT possesses an activity which allows f or displacement synthesis through a minimum of 1,334 bp of duplex DNA- an extent much greater than that required during in vivo reverse trans cription and over 25-fold greater than has been previously demonstrate d for a viral RT. RT does not function as a helicase in the classical sense but appears to closely couple duplex DNA melting with synthesis- driven translocation of the enzyme. In the absence of synthesis, the u nwound region created by a primer-positioned RT appears to be no great er than 2 bp and does not advance along the template. Additionally, RT does not utilize ATP or any deoxynucleoside triphosphate not directly encoded by the template strand to catalyze processive duplex unwindin g at a nick; nor does binding of the enzyme unwind duplex DNA in the a bsence of a 3' terminus. The approximate maximum chain elongation rate during strand displacement synthesis by Moloney murine leukemia virus RT falls between 0.73 and 1.5 nucleotides per s at 37 degrees C. The RNase H activity of RT does not appear to play a role in displacement synthesis; however, a 181-amino-acid C-terminal truncation of RT displ ays a dramatically reduced ability to catalyze synthesis through duple x DNA.