Probing Structure/Function Relationships of HIV-1 Reverse Transcriptase with Styrene Oxide N2-Guanine Adducts

Abstract

Details of the interactions between the human immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA were probed both by introducing sitespecific 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 N 2 -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 ␣-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 ␣-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that ␣-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.During the last 10 years, the use of site-specifically and stereospecifically modified oligonucleotides has played a major role not only in defining which DNA adducts are mutagenic but also in instructing us how different DNA polymerases interact at those sites. We have expanded these studies to use DNA lesions as a probe of polymerase structure and function. The decision to use HIV-1 1 RT as the polymerase of choice is warranted not only because the cocrystal structure is known for an intact enzyme/DNA complex (1) but also because of the tremendous biological significance of RT. Previously using this strategy, we demonstrated that the major groove DNA adducts, N 6 -adenine SO lesions, were readily bypassed by wild type RT, only to experience termination/pause sites 3-5 bases beyond the site of the lesion (2). These sites of enhanced termination probabilities corresponded to the adduct being positioned in the template-primer duplex near the thumb subdomain of the polymerase, where the DNA is in close contact with an ␣-helix and is making the transition from A to B form DNA. Our results could be rationalized because in this bend, the minor groove is widened with a compensatory narrowing of the major groove. The presence of an adduct in the major groove at this position was hypothesized to be the likely source of the enhanced termination through alteration of the minor groove ␣-helix H contacts, 3-5 bases upstream of the 3Ј-OH.To further test whether DNA adducts can give insight into the structure and functioning of HIV-1 RT, we have carried out a study in which monocyclic, minor groove adducts known to cause no significant perturbation to the local ...

Kinetics of Nucleotide Incorporation Opposite DNA Bulky Guanine N2 Adducts by Processive Bacteriophage T7 DNA Polymerase (Exonuclease–) and HIV-1 Reverse Transcriptase

“…Polymerases varied in their abilities to "read through" the adduct (74); interestingly, the mutagenesis studies (in E. coli) were reported not to require SOS function (46). Another interesting observation with RT was that, for both the N 6 -dA and N 2 -dG adducts derived from styrene oxide, RT showed selective pausing at sites several residues beyond the site of incorporation (73,75). Such behavior was not observed in our studies with dG-styrene-R (Fig.…”

“…c Estimated from the the limit of detection at the highest dCTP and dATP concentrations used (2 mM). derived from styrene oxide, both in E. coli cells and with several purified polymerases (46,(73)(74)(75)(76). Polymerases varied in their abilities to "read through" the adduct (74); interestingly, the mutagenesis studies (in E. coli) were reported not to require SOS function (46).…”

Vertical-scanning Mutagenesis of a Critical Tryptophan in the “Minor Groove Binding Track” of HIV-1 Reverse Transcriptase

“…The SO lesions used in this study are known to lie in the major groove without causing a significant perturbation to the local DNA structure (21,22). In contrast, DNA binding by RT is predominantly through the DNA minor groove and RT is unable to bypass R-or S-SO lesions situated in the DNA minor groove (34). However, it is important to note that the sites of termination (positions ϩ1 and ϩ3 in the DNA template) correspond to the major groove adduct being positioned in the template-primer stem in the region of the RT-induced DNA bend.…”

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

“…The N-2 atom of guanine is susceptible to attack by a wide variety of chemicals, including formaldehyde (21), acetaldehyde (22)(23)(24)(25), styrene oxide (26), ␣-hydroxy-N-nitrosopyrrolidine (27), oxidation products of heterocyclic aromatic amines (28), and various polycyclic aromatic hydrocarbons (29,30), leading to the formation of various N 2 -alkyl G adducts. Pyridyloxobutyl-derived N 2 -alkyl G adducts have been detected in rats after chronic treatment with NЈ-nitrosonornicotine (31).…”