Structural alterations in the DNA ahead of the primer terminus during displacement synthesis by reverse transcriptases.
Unlike most DNA polymerases, reverse transcriptases can initiate DNA synthesis at a single-strand break and displace the downstream non- template 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-template strand is similar to what was observed previously for the template strand downstream from the primer terminus. Use of potassium permanganate 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.
Winshell
J
J
Department of Microbiology, School of Medicine University of Washington, Seattle, WA 98195-7242, USA.
Champoux
J J
JJ
D018983:DNA Footprinting; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D005822:Genetic Vectors; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology; D006801:Humans; D009052:Leukemia Virus, Murine / Q000201:enzymology; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011196:Potassium Permanganate / Q000378:metabolism; D013936:Thymidine / Q000737:chemistry
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D011196:Potassium Permanganate; D054303:HIV Reverse Transcriptase; D003850:Deoxyribonuclease I; D013936:Thymidine