Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

Hsu, Gerald W.; Kiefer, James R.; Burnouf, Dominique; Bécherel, Olivier J.; Fuchs, Robert P. P.; Beese, L.S.

doi:10.1074/jbc.m409224200

Cited by 73 publications

(109 citation statements)

References 80 publications

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“…The tertiary structure of DNA polymerase is such that the enzyme fits over the previously formed base pairs 1, 2. These bases must be paired correctly for the polymerase to adopt its functional conformation 3, 4, 5…”

Section: Introductionmentioning

confidence: 99%

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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Pol III core is the three‐subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two‐step reaction scheme in which pol III core binds bimolecularly to the primer‐template junction, then converts at rate k 2 into an exo‐competent conformation. Fits to the force dependence of k init show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature‐ and mismatch‐dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

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Section: Introductionmentioning

confidence: 99%

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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“…The dramatic differences in the rates of nucleotide insertion (~10 4 -fold) observed for dG [FAF]:dC match and dG [FAF]:dA mismatch emphasize the importance of adduct-induced steric constraints in the DNA binding area, especially the minor groove interaction between the template-primer DNA and the polymerase, for determining replication fidelity (52)(53)(54)(55). Altering the adduct-induced conformational equilibria is important, but the complex features of enzyme structure and interaction with the lesion must be taken into consideration.…”

Section: Long-range Conformational Distortion Effectsmentioning

confidence: 99%

“…Hsu et al (52) presented snapshot structures of the AF-adduct docked in the active site of a BF that undergoes one round of replication both in solution and as a crystal. The syn-AF-adduct occupied the preinsertion site with minimal perturbation of the active site.…”

Section: Long-range Conformational Distortion Effectsmentioning

confidence: 99%

Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

Meneni¹,

Liang²,

Cho³

2007

Journal of Molecular Biology

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SUMMARYAdduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2′-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the 'B-DNA' major groove, S is 'stacked' into the helix with base-displacement, and W is 'wedged' into the minor groove. In the present study, we conducted 19 F NMR, CD, Tm, and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B-or S-conformers while the FAF-G:A mismatch ones preferred the B-and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and Tm results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/ translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller and Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.

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“…Three conformations have been characterized in the duplex setting: the major groove external "B type" (B), the base-displaced "stacked" (S), and the minor groove "wedged" (W) conformers ( Figure 1B). Small energy differences among these conformers could shift the adduct population balance at the ss/ds replication fork or in the active site of a polymerase, making it difficult to delineate structure-function relationships (13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

Jain

Reshetnyak²,

Gao³

et al. 2008

Chem. Res. Toxicol.

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Fluorescence spectroscopy was used to study carcinogen-induced conformational heterogeneity in DNA duplexes. The fluorophore 2-aminopurine (AP) was incorporated adjacent (5′) to the lesion (G*) in eight different DNA duplexes [d(5′-CTTCTPG*NCCTC-3′):d(5′-GAGGNXTAGAAG-3′), G* = FAF adduct, P = AP, N = G, A, C, T, and X = C, A] modified by FAF [N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene], a fluorine-tagged model DNA adduct derived from the potent carcinogen 2-aminofluorene. Steady-state measurements showed that fluorescence intensity and Stern-Volmer constants (K sv ) derived from acrylamide quenching experiments decreased for all carcinogen-modified duplexes relative to the controls, which suggests greater AP stacking in the duplex upon adduct formation. Conformation-specific stacking of AP with the neighboring adduct was evidenced by a sequence-dependent variation in fluorescence intensity, position of emission maximum, degree of emission quenching by acrylamide, and temperature-dependent spectral changes. The magnitude of stacking was in the order of FAF residue in base-displaced stacked (S) > minor groove wedged (W) > major groove B type (B). This work represents a novel utility of AP in probing adduct-induced conformational heterogeneities in DNA duplexes.

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Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

Cited by 73 publications

References 80 publications

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

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