Nonpolar thymine isosteres in the Ty3 polypurine tract DNA template modulate processing and provide a model for its recognition by Ty3 reverse transcriptase.
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.
Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Daniela
D
Lener
Mamuka
M
Kvaratskhelia
Stuart F J
SF
Le Grice
D020134:Catalytic Domain; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013941:Thymine / Q000737:chemistry*
D004271:DNA, Fungal; D004279:DNA, Viral; D012331:RNA, Fungal; D012367:RNA, Viral; D018626:Retroelements; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D013941:Thymine