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 synthesis and to characterize the activity with natural viral templates, displacement and non-displacement synthesis were compared under a variety of conditions using the viral long terminal repeat plus strand as the template. Although the rates of both displacement and non-displacement synthesis varied regionally over the template, on the average, displacement synthesis was slower by a factor of approximately 3 to 4. Surprisingly, with one particular primer situated downstream of the tRNA 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 pausing during the two modes of synthesis are different in both nucleotide preference and position relative to the enzyme, suggesting that the enzyme contacts the DNA differently under the two modes of synthesis. It was found that pausing during displacement synthesis did not reflect those local regions of DNA with a predicted high degree of thermal stability. Moreover, the very similar effects of temperature on the rates of displacement and non-displacement synthesis make unlikely a strictly passive mechanism of displacement synthesis whereby breathing of the downstream duplex is sufficient for advancement of the polymerase. Together, these results suggest a mechanism of displacement synthesis in which reverse transcriptase actively participates in the process of strand separation in front of the translocating polymerase.
Whiting
S H
SH
School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
Champoux
J J
JJ
D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry*; D004355:Drug Stability; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013698:Templates, Genetic; D013816:Thermodynamics
D017931:DNA Primers; D004279:DNA, Viral; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase