PROPERTIES OF STRAND DISPLACEMENT SYNTHESIS BY MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASE - MECHANISTIC IMPLICATIONS

Previous results indicated that Moloney murine leukemia virus reverse transcriptase is capable of extensive synthesis under conditions where it must simultaneously displace a downstream non-template DNA strand. To investigate more fully the mechanistic basis for displacement synt hesis and to characterize the activity with natural viral templates, d isplacement and non-displacement synthesis were compared under a varie ty of conditions using the viral long terminal repeat plus strand as t he template. Although the rates of both displacement and non-displacem ent synthesis varied regionally over the template, on the average, dis placement syn thesis was slower by a factor of approximately 3 to 4. S urprisingly, with one particular primer situated downstream of the tRN A primer binding site, displacement synthesis was found to be at least tenfold more processive than non-displacement synthesis, approaching a value of 500 nucleotides. The sequence features associated with paus ing during the two modes of synthesis are different in both nucleotide preference and position relative to the enzyme, suggesting that the e nzyme contacts the DNA differently under the two modes of synthesis. I t was found that pausing during displacement synthesis did not reflect those local regions of DNA with a predicted high degree of thermal st ability. Moreover, the very similar effects of temperature on the rate s of displacement and nondisplacement synthesis make unlikely a strict ly passive mechanism of displacement synthesis whereby breathing of th e downstream duplex is sufficient for advancement of the polymerase. T ogether, these results suggest a mechanism of displacement synthesis i n which reverse transcriptase actively participates in the process of strand separation in front of the translocating polymerase. (C) 1998 A cademic Press Limited.