Probing structure/function relationships of HIV-1 reverse transcriptase with styrene oxide N2-guanine adducts.
Details of the interactions between the human immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA were probed both by introducing site-specific and stereospecific modifications into DNA and by altering the structure of potential critical residues in the polymerase. Unadducted 11-mer DNAs and 11-mer DNAs containing R and S enantiomers of styrene oxide at N2-guanine were ligated with two additional oligonucleotides to create 63-mers that served as templates for HIV-1 reverse transcriptase replication. Oligonucleotides that primed synthesis 5 bases 3' to the adducts could be extended up to 1 base 3' and opposite the lesion. However, when the positions of the 3'-OH of the priming oligonucleotides were placed 1, 2, 3, 4, 5, and 6 bases downstream of the styrene oxide guanine adducts, replication was initiated, only to be blocked after incorporating 4, 5, 6, and 7 bases beyond the lesion. The sites of this adduct-induced termination corresponded to the position of the DNA where alpha-helix H makes contact with the DNA minor groove, 3-5 bases upstream of the growing 3' end. In addition, mutants of the polymerase in alpha-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that alpha-helix H is a sensitive monitor of modifications in the minor groove of newly synthesized template-primer DNA several bases distal to the 3'-OH.
Forgacs
E
E
Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
Latham
G
G
Beard
W A
WA
Prasad
R
R
Bebenek
K
K
Kunkel
T A
TA
Wilson
S H
SH
Lloyd
R S
RS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D004261:DNA Replication; D004852:Epoxy Compounds / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic
D018736:DNA Adducts; D004852:Epoxy Compounds; D006147:Guanine; D004247:DNA; C013690:styrene oxide; D054303:HIV Reverse Transcriptase