Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate 1 1Edited by J. Karn

Wöhrl, Birgitta M.; Krebs, Ruth; Goody, Roger S.; Restle, Tobias

doi:10.1006/jmbi.1999.3057

Cited by 72 publications

(92 citation statements)

References 44 publications

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“…4 are consistent with a model in which the second complex, Ib, must first undergo an isomerization step before nucleotide incorporation can occur and would represent a ''slipped'' structure in which the dp͞dt is bound in the correct orientation but is not correctly positioned in the binding cleft for nucleotide binding (8). An attractive possibility is that the 3Ј end of the primer has moved further into the polymerase active site, so that the terminal nucleotide is now occupying the binding site for incoming nucleoside triphosphate.…”

Section: Resultssupporting

confidence: 83%

“…As sketched in Fig. 4, we suggest that the first and major complex with a shot-noise limited R DA -distribution, which we refer to as the productive complex in its educt state (Ia: P-E), is capable of facile incorporation of nucleotides at a rate constant of Ϸ50 s Ϫ1 , as measured previously (8), and probably corresponds to the structure seen in the x-ray crystallographic analysis of Huang et al (6). The second complex, species Ib, is characterized by a broad R DA distribution in which the mean distance between the dyes has shortened by ϳ5 Å (Fig.…”

Section: Resultssupporting

confidence: 67%

“…The actual single molecule measurements confirmed that the affinity was high enough to ensure complex formation, because most RT molecules sampled contained bound (labeled) substrate. Ensemble measurements on single nucleotide incorporation under these salt conditions showed similar kinetic behavior to that seen at higher salt, with three kinetic phases being observed (8).…”

mentioning

confidence: 56%

“…4. According to this interpretation, the slower of the two fast phases seen in the nucleotide incorporation kinetics (8) corresponds to this movement, which is an essential step that must occur after nucleotide incorporation and represents the fundamental translocation step in the polymerization reaction. This step seems to be correlated with the previously reported phase occurring at Ϸ2 s Ϫ1 (8), after which binding of the next nucleotide and incorporation occurs at Ϸ50 s Ϫ1 , the rate constant of the fastest phase seen in the kinetic experiments (8).…”

Section: Resultsmentioning

confidence: 99%

“…To date, these crystallographic models have indicated a single p͞t-binding mode. In contrast, recent solutionbased kinetic studies on p͞t binding and nucleotide incorporation by RT suggest a heterogeneous mixture of several different binding modes (8). To confirm directly the existence of several species, and to obtain structural and functional information about each, we have used single-molecule spectroscopy to investigate RT:p͞t complexes in solution.…”

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confidence: 99%

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Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Rothwell

Berger

Kensch

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

217

244

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By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.H IV-1 reverse transcriptase (RT) is a key enzyme in the life cycle of HIV. This multifunctional enzyme is responsible for the complex process of transcribing viral RNA into doublestranded DNA for integration into the host cell genome. The enzyme is a heterodimer composed of subunits which share a common N terminus and have subdomains referred to as fingers, palm, thumb, and connection ( Fig. 1). Although the subdomains of each subunit are structurally similar, the overall folding within the two subunits is quite different (1). The large subunit, p66, contains RNA-and DNA-dependent DNA polymerase as well as RNase H activities. The p51 subunit, which is inactive in the heterodimer but active in homodimers (2, 3), is thought to play a role in stabilizing the conformation of p66.Crystallographic studies of RT:nucleic acid primer͞template (p͞t) complexes have provided valuable insights into the structural features and conformational changes induced by p͞t binding (1, 4-7). To date, these crystallographic models have indicated a single p͞t-binding mode. In contrast, recent solutionbased kinetic studies on p͞t binding and nucleotide incorporation by RT suggest a heterogeneous mixture of several different binding modes (8). To confirm directly the existence of several species, and to obtain structural and functional information about each, we have used single-molecule spectroscopy to investigate RT:p͞t complexes in solution.Single-molecule techniques have proven to be valuable tools for resolving static and dynamic heterogeneity of an ensemble (9-13). For this investigation, we use a newly developed multiparameter fluorescence detection (MF...

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Section: Resultssupporting

confidence: 83%

Section: Resultssupporting

confidence: 67%

mentioning

confidence: 56%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Rothwell

Berger

Kensch

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

217

244

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Varied Active‐Site Constraints in the Klenow Fragment of E. coli DNA Polymerase I and the Lesion‐Bypass Dbh DNA Polymerase

et al. 2008

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We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.

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Double Cyclization of Bis(α‐hetarylmethyl)amino Esters to Optically Active Bridged N‐Heterocycles of HIV‐Inhibiting Activity

et al. 2004

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Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate 1 1Edited by J. Karn

Cited by 72 publications

References 44 publications

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Varied Active‐Site Constraints in the Klenow Fragment of E. coli DNA Polymerase I and the Lesion‐Bypass Dbh DNA Polymerase

Double Cyclization of Bis(α‐hetarylmethyl)amino Esters to Optically Active Bridged N‐Heterocycles of HIV‐Inhibiting Activity

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