Reference: Lener D, et al. (2003) Nonpolar thymine isosteres in the Ty3 polypurine tract DNA template modulate processing and provide a model for its recognition by Ty3 reverse transcriptase. J Biol Chem 278(29):26526-32

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Abstract


Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (-)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T --> F substitution and enhanced ribonuclease H (RNase H) activity approximately 12-13 bp downstream. In the most pronounced case, dual T --> F substitution at PPT positions -1/-2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (-)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT approximately 12-13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T --> F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5' vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper.

Reference Type
Journal Article | Comparative Study
Authors
Lener D, Kvaratskhelia M, Le Grice SF
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