The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of the HIV-1 virus in the absence of viral infectivity factor (Vif). The molecular mechanism of A3G antiviral activity is primarily ...attributed to deamination of single-stranded DNA (ssDNA); however, the nondeamination mechanism also contributes to HIV-1 restriction. The interaction of A3G with ssDNA and RNA is required for its antiviral activity. Here we used atomic force microscopy to directly visualize A3G–RNA and A3G–ssDNA complexes and compare them to each other. Our results showed that A3G in A3G–RNA complexes exists primarily in monomeric–dimeric states, similar to its stoichiometry in complexes with ssDNA. New A3G–RNA complexes in which A3G binds to two RNA molecules were identified. These data suggest the existence of two separate RNA binding sites on A3G. Such complexes were not observed with ssDNA substrates. Time-lapse high-speed atomic force microscopy was applied to characterize the dynamics of the complexes. The data revealed that the two RNA binding sites have different affinities for A3G. On the basis of the obtained results, a model for the interaction of A3G with RNA is proposed.
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The DNA base excision repair (BER) pathway is essential for maintaining genomic integrity and is implicated in active DNA demethylation, a key element of epigenetic transcriptional ...regulation. Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs, initiating repair of deaminated 5‐methylcytosine (mC). TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidation products of mC produced by Tet enzymes. These seemingly disparate activities are consistent with TDG specificity for acting at CpG sites and its essential role in active DNA demethylation and embryonic development. Understanding how glycosylases excise lesions and avoid acting on undamaged DNA is an important problem in DNA repair. Structural and biochemical results here reveal how TDG attains broad specificity for G·T and G·fC lesions while avoiding A·T pairs. A crystal structure of TDG (catalytic domain) bound to substrate analogue suggests G·T glycosylase activity is suboptimal owing to unfavorable interactions between flipped dT substrate and two active‐site residues. Remarkably, mutating these residues greatly increases G·T activity and confers substantial activity for normal A·T base pairs. The results suggest TDG evolved with suboptimal G·T repair capability in order to minimize aberrant activity on undamaged DNA, an unprecedented finding for a repair enzyme. Supported by NIH (R01‐GM072711).
Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and ...follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G · U activity, but its role in G · T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G · U mispairs and abasic (G · AP) sites, and somewhat less tightly G · T mispairs. Kinetics experiments show 1:1 binding provides full G · T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G · T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G · U or G · T lesions.
Thymine DNA glycosylase (TDG) promotes genomic integrity by removing thymine from mutagenic G·T mispairs arising from deamination of 5‐methylcytosine and follow‐on base excision repair enzymes ...restore a G·C pair. Previous studies suggested that Asn140, a strictly conserved active site residue is important, for base excision, yet its catalytic role had not been investigated rigorously. To further our understanding of the catalytic mechanism of TDG, here we determined the contribution of Asn140 to the substrate binding and chemical steps of the reaction. Isothermal titration calorimetry (ITC) experiments show that TDG‐N140A variant binds substrate analogs with the same tight affinity as wild‐type TDG, indicating Asn140 does not contribute to substrate binding. Single turnover kinetics experiments show that TDG‐N140A exhibits no detectable base excision activity for a G·T substrate, and its excision rate is vastly diminished (by ~104.4fold) for G·U, G·FU, and G·BrU substrates. Our findings indicate that Asn140 is essential for the chemical step, but does not contribute substantially to substrate binding. Thus N140A variant provides a useful platform for investigating the role of other residues in forming the reactive enzyme‐substrate complex. This work was supported by an NIH grant.
Repair of GADTT mismatches arising from deamination of 5-methylcytosine (m5C) involves excision of thymine and restoration of a GADTC pair via base excision repair (BER). Thymine DNA glycosylase ...(TDG) is one of two mammalian enzymes that can specifically remove thymine from GADTT mispairs. While TDG can excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m5C is an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps to maintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation, which may involve excision of actively deaminated m5C. The relatively weak GADTT activity of TDG has been implicated in the hypermutability of CpG sites, which largely involves CaT transitions arising from m5C deamination. Thus, it is important to understand how TDG recognizes and process substrates, particularly GADTT mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substrate binding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant for pH 5.5a9, but falls sharply for pH>9 due to severely weakened substrate binding, and, potentially, ionization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength, particularly for GADTT substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and irreversibly at 37ADGC, but can be stabilized by specific and nonspecific DNA. The temperature dependence of catalysis reveals large and unexpected differences for GADTU and GADTT substrates, where GADTT activity exhibits much steeper temperature dependence. The results suggest that reversible nucleotide flipping is much more rapid for GADTT substrates, consistent with our previous findings that steric effects limit the active-site lifetime of thymine, which may account for the relatively weak GADTT activity. Our findings provide important insight into catalysis by TDG, particularly for mutagenic GADTT mispairs.
Deamination of 5‐methylcytosine produces G·T mispairs, a mutagenic event that contributes to cancer and genetic disease. Thymine DNA glycosylase (TDG) recognizes these G·T lesions and excises ...thymine, and downstream base excision repair (BER) proteins restore a G·C pair. The task of excising a normal base from a mismatch, while avoiding the huge excess of undamaged DNA, presents a formidable challenge for a DNA glycosylase. TDG can also remove other damaged bases, including uracil and 5‐halogenated uracils, and has specificity for excising bases paired with guanine and located in a CpG context. We solved a crystal structure of TDG (catalytic domain) bound to DNA with a nonhydrolyzable substrate analog (2′‐fluoroarabino‐dU) flipped into its active site, providing a snapshot of the enzyme‐substrate complex in the absence of base‐sugar bond cleavage. TDG forms interactions with the uracil base that likely stabilize nucleotide flipping and promote specificity. The structure suggests that steric interactions with the methyl group of thymine could explain in part the much weaker binding observed for G·T relative to G·U substrates. Such interactions could potentially serve to minimize the aberrant excision of T from A·T pairs. Our structure indicates the mechanism of substrate recognition differs substantially for TDG enzymes relative to the closely related MUG enzymes. Supported by a grant from the NIH (GM‐072711).
Development of small synthetic transcription factors is important for future cellular engineering and therapeutics. This article describes the chemical synthesis of α-amino-isobutyric acid (Aib) ...substituted, conformationally constrained, helical peptide mimics of Cro protein from bacteriophage λ that encompasses the DNA recognition elements. The Aib substituted constrained helical peptide monomer shows a moderately reduced dissociation constant compared to the corresponding unsubstituted wild type peptide. A suitably cross-linked dimeric version of the peptide, mimicking the dimeric protein, recapitulates some of the important features of Cro. It binds to the operator site OR3, a high affinity Cro binding site in the λ genome, with good affinity and single base-pair discrimination specificity. A dimeric version of an even shorter peptide mimic spanning only the recognition helix of the helix-turn-helix motif of the Cro protein was created following the same design principles. This dimeric peptide binds to OR3 with affinity greater than that of the longer version. Chemical shift perturbation experiments show that the binding mode of this peptide dimer to the cognate operator site sequence is similar to the wild type Cro protein. A Green Fluorescent Protein based reporter assay in vivo reveals that the peptide dimer binds the operator site sequences with considerable selectivity and inhibits gene expression. Peptide mimics designed in this way may provide a future framework for creating effective synthetic transcription factors.
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Thymine DNA glycosylase (TDG) is a base excision repair enzyme that excises T from G·T mispairs and removes other lesions, exhibiting specificity for damage at CpG sites. DNA ...glycosylases use nucleotide flipping to find lesions and cleave the base‐sugar bond. Typically, a bulky side chain penetrates the DNA, filling the space vacated by the flipped nucleotide. Our crystal structure of human TDG catalytic domain bound to abasic DNA shows that an arginine (R275) plugs the helical gap and contacts two phosphates flanking the abasic sugar (Maiti A,
et al
.,
Proc Natl Acad Sci 105
: 8890‐8895). Here, we examine the role of R275 in the catalytic mechanism of TDG. Isothermal titration calorimetry (ITC) experiments show that substrate (analog) binding affinity is significantly lower for R275A‐hTDG versus hTDG, and lower still for R275L‐hTDG. Single turnover kinetics experiments show that k
max
is significantly lower for R275A‐hTDG versus hTDG, and lower still for R275L‐hTDG. Our results are surprising, because two related enzymes, mismatch‐specific uracil glycosylase and uracil DNA glycosylase have leucine at the position corresponding to R275 of hTDG. We find that mutation of R275 has no effect on steady‐state turnover, k
cat
, suggesting that R275 cannot account for the exceedingly slow product release observed for hTDG. This work was supported by an NIH grant (to ACD).
Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. ...TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (kobs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of kobs on TDG concentration gives kmax = 1.6 min−1 and K1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (Kd ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.
Post-translational SUMO modification of TDG weakens its DNA binding and was proposed to regulate dissociation of a tight enzyme-product complex.
In vitro sumoylation of TDG by SUMO-1 and SUMO-2 is efficient for free and DNA-bound TDG.
E2-mediated sumoylation is not selective for product-bound TDG but could potentially stimulate product release.
Our findings inform the mechanism and role of TDG sumoylation.
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Base excision repair (BER) is essential for maintaining genetic integrity and for active DNA demethylation, a central element of epigenetic gene regulation. A key player in both ...processes is thymine DNA glycosylase (TDG), which excises modified forms of 5‐methylcytosine (mC). TDG excises thymine from mutagenic G/T mispairs arising from mC deamination. TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidized forms of mC generated by Tet enzymes. TDG is essential for embryonic development, reflecting a crucial function in regulating gene expression that likely involves a key step in active DNA demethylation. We used structural, biochemical, and cell‐based methods to understand how TDG attains specificity for excising select forms of modified mC (i.e., T, fC, and caC). We identified a TDG variant that retains fC activity, but lacks detectible caC activity, indicating a fundamental difference in the excision mechanism for these related bases. We used this variant to study the role of TDG in active DNA demethylation in human cells. The results indicate that TDG excision of fC can account for findings that caC is depleted in cells expressing TDG, consistent with the fact that fC is a precursor for Tet‐mediated formation of caC. The results suggest that TDG excision of fC could be a predominant element in a pathway for active DNA demethylation. Supported by the NIH (R01‐GM072711).
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