Deoxynucleotide polymerization by HIV-1 reverse transcriptase is terminated by site-specific styrene oxide adducts after translesion synthesis.
In an effort to integrate an understanding of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) structure with its function, the action of HIV-1 RT was examined in vitro on DNA templates modified with a model bulky DNA adduct. Styrene oxide was site-specifically and stereospecifically coupled to the N6 position of adenine to form six different adducted templates. Primer extension assays were conducted under conditions defining both single and multiple encounters between the polymerase and the damaged template-primer. The extent of polymerization observed for each adduct was found to depend on both the chirality of the damage and the lesion sequence context. When HIV-1 RT polymerization was limited by single encounters with damaged DNA, the SO lesions were readily bypassed as evidenced by minimal pausing at the adducted base. However, RT replication of all SO-modified templates was significantly terminated 3-5 nucleotides after translesion synthesis but before reaching the end of the template. These truncated products could be readily extended when additional encounters between enzyme and template-primer were permitted. A model is presented to explain these results in the context of HIV-1 RT structure during DNA replication.
Latham
G J
GJ
Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235.
Lloyd
R S
RS
D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D002384:Catalysis; D018736:DNA Adducts; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism*; D004852:Epoxy Compounds / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic
D001704:Biopolymers; D018736:DNA Adducts; D004279:DNA, Viral; D004852:Epoxy Compounds; C013690:styrene oxide; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase