Structural alterations in the DNA ahead of the primer terminus duringdisplacement synthesis by reverse transcriptases

摘要

Unlike most DNA polymerases, reverse transcriptases can initiate DNA synthesis at a single-strand break and displace the downstream nontemplate strand simultaneously with extension of the primer. This reaction is important for generation of the long terminal repeat sequences in the duplex DNA product of retroviral reverse transcription. Oligonucleotide-based model displacement constructs were used to study the interaction of human immunodeficiency virus type 1 and Moloney murine leukemia virus reverse transcriptases with the DNA. Under conditions where the DNA is saturated with enzyme, there is no protection against DNase I cleavage of the 5' single-stranded extension that would correspond to the already-displaced strand. However, the DNase I footprint on the non-template strand extends from the +1 to the +9 position for the human immunodeficiency virus type 1 enzyme and from +1 to +7 or +8 for the Moloney enzyme. This extent of protection on the non-tem plate strand is similar to what was observed previously for the template strand downstream from the primer terminus. Use of potassium per manganate as a probe for unpaired bases in the region ahead of the primer terminus reveals that the two base-pairs immediately in front of the enzyme are melted by the bound enzyme. These findings are consistent with a displacement mechanism in which the reverse transcriptase plays an active role in unpairing the DNA ahead of the translocating polymerase. The results are interpreted in light of a recent crystal structure showing the nature of the protein-DNA contacts with the template strand ahead of the primer terminus.