Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 May 30;289(22):15810-9.
doi: 10.1074/jbc.M114.572081. Epub 2014 Apr 21.

E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex

Christopher T Coey  1 , Megan E Fitzgerald  1 , Atanu Maiti  1 , Katherine H Reiter  2 , Catherine M Guzzo  2 , Michael J Matunis  2 , Alexander C Drohat  3
Affiliations

E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex

Christopher T Coey et al. J Biol Chem. .

Abstract

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.

Keywords: Base Excision Repair (BER); DNA Methylation; DNA Repair; Enzyme Turnover; Post-translational Modification (PTM); Small Ubiquitin-like Modifier (SUMO); Sumoylation; Ubiquitin-conjugating Enzyme (E2 Enzyme).

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Putative mechanism by which sumoylation of TDG suppresses binding to DNA. Because a crystal structure has not been solved for sumoylated TDG bound to DNA, we show a model of this complex to illustrate how sumoylation likely suppresses DNA binding. The model was generated by aligning the structure of sumoylated TDG (PDB ID code 1WYW) with the structure of TDG bound to abasic DNA (PDB ID code 2RBA); only sumoylated TDG is shown. The graphic depiction of full-length TDG shows structured regions as rectangles (colored to correspond with the model below), whereas unstructured regions are depicted as lines. The catalytic core (117–300) and C-terminal region (301–332) of human TDG are colored cyan and blue, respectively, and SUMO-1 is colored magenta. The β-strand and α-helix in the unstructured C-terminal region of TDG (blue) are thought to be stabilized by SUMO-1, and the helix would likely form a steric clash with DNA (circled in blue). The abasic site is flipped into the active site of the catalytic domain (circled in red).
FIGURE 2.
FIGURE 2.
Sumoylation of RanGAP1 and TDG at 22 °C. A and B, in vitro sumoylation experiments performed for modification of RanGAP1-NΔ419 and TDG by SUMO-1 (A) or SUMO-2 (B) and monitored by electrophoresis. The first lane of each gel contains pure TDG, and the second lane contains pure SUMO-1-modified TDG (TDG∼SUMO-1 or TDG-S). Reactions were initiated by adding ATP (2.5 mm) to buffer containing E1 (0.1 μm), E2 (1 μm), SUMO-1 or SUMO-2 (10 μm), and either RanGAP1-NΔ419 or TDG (2 μm). C, in vitro sumoylation of TDG with SUMO-2 over a 24-h time course. Samples were extracted at the time points given. D, in vitro sumoylation of TDG with SUMO-1 and SUMO-2 using SUMO concentrations of <10 μm. SUMO concentrations for a given assay are listed above each set of time points. Samples were extracted from the reaction and quenched at the indicated times (given in minutes), and the reaction progress was analyzed by SDS-PAGE under reducing conditions.
FIGURE 3.
FIGURE 3.
Sumoylation of TDG at 37 °C in the presence and absence of DNA. A–C, in vitro sumoylation reactions for modification of TDG by SUMO-1 or SUMO-2 performed at 37 °C in the absence of DNA (A) or in the presence of abasic DNA (B) or nonspecific (undamaged) DNA (C), and monitored by electrophoresis. The first lane of each gel contains pure TDG, and the second lane contains pure SUMO-1-modified TDG (TDG∼SUMO-1 or TDG-S). The sumoylation reactions were initiated by adding ATP (2.5 mm) to buffer containing E1 (0.1 μm), E2 (1 μm), SUMO-1 or SUMO-2 (10 μm), TDG (2 μm), with no DNA or with abasic DNA (2.5 μm) or nonspecific DNA (4.1 μm). D, in vitro sumoylation of RanGAP1-NΔ419 at 37 °C. Samples were extracted from the reaction and quenched at the indicated times (given in minutes), and the reaction progress was analyzed by SDS-PAGE under reducing conditions.
FIGURE 4.
FIGURE 4.
Sumoylation of free and DNA-bound TDG in the presence and absence of HeLa nuclear extract. A–C, immunoblot analysis of TDG modification in the presence and absence of nuclear extract for free TDG (A), TDG bound to nonspecific DNA (B), and TDG bound to AP-DNA (C). Reactions were performed in sumoylation buffer A with 0.1 μm E1, 0.5 μm E2, 0.8 μm TDG, and 5 μm SUMO-1 and were initiated by the addition of 10 mm ATP. As indicated, some reactions contained 15 μg of HeLa NE and/or 5 μm S2A (a SENP inhibitor). Samples were extracted from the reaction at the indicated time points, diluted 10-fold into buffer A, and quenched with 5×sample buffer before separation via SDS-PAGE under reducing conditions and transfer to nitrocellulose membranes. TDG was detected via immunoblotting with anti-His antibody and a fluorescent secondary antibody.
FIGURE 5.
FIGURE 5.
Single-turnover assays of TDG modification by preloaded E2∼SUMO-1. A, immunoblot analysis of TDG modification by preformed E2∼SUMO-1 in the presence or absence of DNA. Preformed E2∼SUMO-1 conjugate was generated by adding ATP (10 μm) to buffer containing E1 (0.1 μm), E2 (1 μm), and SUMO-1 (1 μm). Reactions were incubated for 15 min at 37 °C. Then, TDG modification reactions were initiated by adding 3 μl of the E2∼SUMO-1 reaction to 27 μl of buffer containing TDG (2 μm) and EDTA (5 mm). Samples were extracted from the reaction and quenched at the indicated times (given in seconds), and the formation of TDG∼SUMO-1 was analyzed by SDS-PAGE under nonreducing conditions. Detection of SUMO-1 conjugates was performed via immunoblotting with anti-SUMO-1 primary antibody, followed by blotting with a fluorescent secondary antibody. B, kinetics for modification of TDG by preformed E2∼SUMO-1 in the presence or absence of DNA. Linear regression analysis provides initial rate constants of kobs = 1.1 min−1 (no DNA), 0.78 min−1 (AP-DNA), and 0.53 min−1 (NS-DNA). Error bars represent 1 S.D. The asterisk indicates the 60-s time point for free TDG, which was not used in data fitting.
FIGURE 6.
FIGURE 6.
Dependence of modification rate on TDG concentration. Immunoblot analysis of TDG modification by preformed E2∼SUMO-1 was performed in the absence of DNA at TDG concentrations of 0.12 μm, 0.23 μm, 0.45 μm, 0.90 μm, 1.8 μm, and 3.6 μm. The kobs values were obtained by linear regression analysis of at least three time points per experiment. Error bars represent 1 S.D. Where error bars are not visible, they are smaller than the symbols. Data were fitted to a hyperbolic equation, kobs = kmax[TDG]/(K1/2 + [TDG]). The fitting yields kmax = 1.55 ± 0.16 min−1 and K1/2 = 0.55 ± 0.17 μm.
FIGURE 7.
FIGURE 7.
EMSA analysis of AP-DNA binding by unmodified TDG and TDG∼SUMO-1. A, electrophoretic mobility assays (EMSAs) performed with 10 nm AP-DNA and varying concentrations (5–500 nm) of unmodified TDG (left panel), in vitro modified TDG (center panel), and recombinant TDG∼SUMO-1 (right panel). The dotted line below the TDG∼SUMO-1 lanes aligns with the midpoint of TDG-bound DNA in a 1:1 complex, indicating a slight, but clear, reduction in mobility for DNA bound to TDG∼SUMO-1 compared with unmodified TDG. B, SDS-PAGE analysis of an overnight in vitro sumoylation reaction performed to obtain the 100% modified TDG∼SUMO-1 used in the EMSA shown in A, center panel.
FIGURE 8.
FIGURE 8.
Summary of findings from these and previous studies regarding TDG sumoylation. A, our results showed that E2∼SUMO efficiently modifies free and DNA-bound TDG, exhibiting no substantial specificity for product-bound TDG. Thus, the previous proposal that TDG is selectively sumoylated when it is bound to AP-DNA (just after base excision) would require an unidentified E3 or other selectivity factor (16). Nevertheless, modification of product-bound TDG by E2∼SUMO is relatively efficient and could potentially stimulate dissociation of the product complex (in the absence of an E3), thereby enhancing enzymatic turnover. Given our finding that TDG∼SUMO-1 still binds AP-DNA with high affinity, it is possible that TDG∼SUMO-1 shuttles on and off of AP-DNA (or undamaged DNA), rather than dissociating completely (as indicated by dotted lines and question mark). However, because TDG∼SUMO lacks G·T activity and E2 modifies free and DNA-bound TDG, efficient desumoylation (by SENPs) is needed to maintain a pool of catalytically active TDG. B, sumoylation of free TDG can modulate its interactions with other proteins (P), which can depend on whether they contain a SIM or are themselves sumoylated. Crystallographic studies (17, 18) indicate that for sumoylated TDG, the covalently tethered SUMO domain occupies its own SIM, which could potentially suppress interactions with a sumoylated partner protein, unless it also contains a SIM. Potential SUMO-mediated interactions of free and modified TDG with sumoylated and/or SIM-containing partners are shown. Checkmarks (in green) indicate allowed SUMO/SIM interactions, question marks indicate interactions that would require a change in SUMO conformation to occur, and crosses (in red) indicate SUMO/SIM-mediated interactions that should not occur.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Neddermann P., Jiricny J. (1993) The purification of a mismatch-specific thymine-DNA glycosylase from HeLa cells. J. Biol. Chem. 268, 21218–21224 - PubMed
    1. Neddermann P., Gallinari P., Lettieri T., Schmid D., Truong O., Hsuan J. J., Wiebauer K., Jiricny J. (1996) Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase. J. Biol. Chem. 271, 12767–12774 - PubMed
    1. Cortellino S., Xu J., Sannai M., Moore R., Caretti E., Cigliano A., Le Coz M., Devarajan K., Wessels A., Soprano D., Abramowitz L. K., Bartolomei M. S., Rambow F., Bassi M. R., Bruno T., Fanciulli M., Renner C., Klein-Szanto A. J., Matsumoto Y., Kobi D., Davidson I., Alberti C., Larue L., Bellacosa A. (2011) Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell 146, 67–79 - PMC - PubMed
    1. Cortázar D., Kunz C., Selfridge J., Lettieri T., Saito Y., MacDougall E., Wirz A., Schuermann D., Jacobs A. L., Siegrist F., Steinacher R., Jiricny J., Bird A., Schär P. (2011) Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature 470, 419–423 - PubMed
    1. Maiti A., Drohat A. C. (2011) Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J. Biol. Chem. 286, 35334–35338 - PMC - PubMed

Publication types

MeSH terms