TY - JOUR
T1 - Probing structure/function relationships of HIV-1 reverse transcriptase with styrene oxide N2-guanine adducts
AU - Forgacs, Eva
AU - Latham, Gary
AU - Beard, William A.
AU - Prasad, Rajendra
AU - Bebenek, Katarzyna
AU - Kunkel, Thomas A.
AU - Wilson, Samuel H.
AU - Lloyd, R. Stephen
PY - 1997/3/28
Y1 - 1997/3/28
N2 - 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 α-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 α-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that α-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.
AB - 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 α-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 α-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that α-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.
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U2 - 10.1074/jbc.272.13.8525
DO - 10.1074/jbc.272.13.8525
M3 - Article
C2 - 9079681
AN - SCOPUS:0030887461
SN - 0021-9258
VL - 272
SP - 8525
EP - 8530
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 13
ER -