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
. 2024 Oct 25;15(1):9226.
doi: 10.1038/s41467-024-53497-7.

Thymine DNA glycosylase combines sliding, hopping, and nucleosome interactions to efficiently search for 5-formylcytosine

Brittani L Schnable  1   2 , Matthew A Schaich  2   3 , Vera Roginskaya  2   3 , Liam P Leary  2   3 , Tyler M Weaver  4 , Bret D Freudenthal  4 , Alexander C Drohat  5 , Bennett Van Houten  6   7   8
Affiliations

Thymine DNA glycosylase combines sliding, hopping, and nucleosome interactions to efficiently search for 5-formylcytosine

Brittani L Schnable et al. Nat Commun. .

Abstract

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylcytosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we study TDG in the presence and absence of 5-formylcytosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of base modifications. TDG active site variants and truncated N-terminus, reveals these variants alter base modification search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG is found to either bypass, colocalize with, or encounter but not bypass the nucleosome. Truncating the N-terminus reduces the number of interactions with the nucleosome. Our findings provide mechanistic insights into how TDG searches for modified DNA bases in chromatin.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. TDG binds DNA specifically and nonspecifically.
a Cartoon schematic showing how 5fC was incorporated into nick translated λ DNA. Created in BioRender. Schnable, B. (2023) BioRender.com/q21t266. b Cartoon schematic showing 28 base pair duplex DNA containing a single 5fC (orange) ligated into 6 kb LUMICKS handle kit, with handle 2 containing ATTO 488 (blue). Created in BioRender. Schnable, B. (2024) BioRender.com/z84c645. c A diagram depicting the order of reagents, which are under laminar flow, are captured in the flowcell. Created in BioRender. Schnable, B. (2023) BioRender.com/k78o196. d A cartoon depiction of the DNA substrate used for TDG binding, with 5fC sites shown in blue, and an example kymograph with TDG binding shown in red. Specific event indicated with gray asterisk and nonspecific event indicated with teal asterisk. e Cumulative Resident Time Distribution (CRTD) analysis fit to a two-phase decay of TDG binding DNA containing 5fC specifically (n = 70) and nonspecifically (n = 487). Data represents the mean ± SEM from four independent experiments. f A cartoon depiction of the unmodified DNA substrate with an example kymograph of TDG binding and moving. g CRTD analysis fit to a one-phase decay of TDG binding unmodified λ DNA (n = 155). Data represents the mean ± SEM of the fit from three independent experiments. h An example kymograph of TDG binding to a single 5fC. i CRTD analysis fit to a two-phase decay of TDG binding to 5fC (n = 28). Data represents the mean ± SEM of the fit from three independent experiments.
Fig. 2
Fig. 2. TDG exhibits linear diffusion on DNA.
a Scatter plot of the diffusion coefficient (log10D) calculated for TDG with increasing ionic strength on unmodified λ. Dashed line, Dlim, theoretical limit to free diffusion for TDG-HaloTag (75 mM NaCl n = 105; 100 mM NaCl n = 101; 150 mM NaCl n = 102). P-values determined by two-way ANOVA. Data represents the mean ± SD from three independent experiments. b An example kymograph of the two-color TDG experiment. Separate TDG-HaloTag extracts were labeled with either JF635 (red) or JF552 (green) and mixed at a 1:1 ratio. White arrows indicate bypass events and yellow arrows indicate collision events.
Fig. 3
Fig. 3. TDG catalytic variants indicate R275 is essential for base detection.
Experiments conducted with unmodified or nick translated DNA containing 5fC. a CRTD analysis fit to a one-phase decay of N140A TDG binding to unmodified (n = 163), specifically to 5fC (n = 30) and nonspecifically (n = 168). b CRTD analysis fit to a one-phase decay of R275A TDG binding to unmodified (n = 110), specifically to 5fC (n = 24) and nonspecifically (n = 296). c CRTD analysis fit to a one-phase decay of R275L TDG binding to unmodified (n = 269), specifically to 5fC (n = 42) and nonspecifically (n = 330). d Scatter plot of the diffusion coefficient (log10D) calculated for each variant TDG on unmodified λ (WT n = 101; N140A n = 106; R275A n = 106; R275L n = 102). Dashed line, Dlim, theoretical limit to free diffusion for TDG-HaloTag. P-values determined by two-way ANOVA. Data represents the mean ± SD from three independent experiments.
Fig. 4
Fig. 4. The N-terminus of TDG is significant for its movement on DNA.
Tension alters diffusivity and α for ΔN-term and full-length TDG. a A cartoon depiction of the DNA substrate with 5fC sites shown in blue, and a representative kymograph with TDG binding shown in red. Break in kymograph to show location of fiducial markers. b CRTD analysis fit to a two-phase decay of TDG binding DNA containing 5fC specifically (n = 32). CRTD analysis fit to a one-phase decay of TDG binding nonspecifically (n = 243). c CRTD analysis fit to a one-phase decay of full-length TDG binding to unmodified DNA at 5 pN (n = 172), 10 pN (n = 27), 20 pN (n = 53), 30 pN (n = 169), and 40 pN (n = 102). d CRTD analysis fit to a one-phase decay of ΔN-term TDG binding to unmodified DNA at 5 pN (n = 113), 10 pN (n = 201), 20 pN (n = 197), 30 pN (n = 293), and 40 pN (n = 203). Data in (b–d) represents the mean ± SEM of the fit from 3 independent experiments. e Plot of diffusion coefficients (D) versus alpha (α) for full-length for 5 pN (n = 50), 10 pN (n = 41), 20 pN (n = 41), 30 pN (n = 57), and 40 pN (n = 33). f Plot of diffusion coefficients (D) versus alpha (α) for ΔN-term for 5 pN (n = 30), 10 pN (n = 36), 20 pN (n = 24), 30 pN (n = 22), and 40 pN (n = 35). g Plot of diffusion coefficients (D) versus alpha (α) for N140A for 10 pN (n = 33), and 40 pN (n = 34). h Plot of diffusion coefficients (D) versus alpha for 10 pN (n = 19) and 40 pN (n = 27). (α) for R275L. i Plot of diffusion coefficients (D) versus alpha (α) for R275A for 10 pN (n = 14) and 40 pN (n = 19). Data in (e–i) represents the mean ± SD from three independent experiments.
Fig. 5
Fig. 5. N-terminus of TDG is important for interacting with nucleosomes.
A cartoon depiction of the DNA substrate with Cy3 labeled nucleosome. Representative kymograph with full-length TDG (shown in red) (a) bypassing, (b) does not bypass, or (c) colocalizes with the nucleosome shown in green. (d) Representative kymograph of ΔN-term TDG (red) colocalizing with nucleosome (green). e CRTD analysis fit to a one-phase decay of TDG interacting with the NCP (n = 28). f Stacked bar graph showing the fraction of bypass (green), no bypass (gray), and colocalized (teal) events for full-length and ΔN-term (p = 0.0011 by Χ2).
Fig. 6
Fig. 6. Working model.
TDG uses multiple modes of linear diffusion to efficiently search for 5fC. a WT TDG (gray) fused to HaloTag (red) scans dsDNA with sliding and hopping interactions, with a lifetime of 7.9 ± 0.06 s and diffusivity of 0.028 ± 0.02 µm2/s. Upon 5fC engagement (orange), TDG binds with a lifetime of 72.9 ± 5.4 s. These interactions are dependent on active site residues N140, R275, and the N-terminal domain. b When TDG encounters a nucleosome core particle, it can bypass, not bypass, or colocalize with the NCP (16.5 ± 1.9 s). Importantly the N-terminal domain of TDG stabilizes the interaction with the NCP, resulting in a four-fold increase in colocalization frequency. Created in BioRender. Schaich, M. (2024) BioRender.com/y41i875.
See this image and copyright information in PMC

Update of

Similar articles

See all similar articles

References

    1. Whitaker, A. M., Schaich, M. A., Smith, M. R., Flynn, T. S. & Freudenthal, B. D. Base excision repair of oxidative DNA damage: from mechanism to disease. Front. Biosci. (Landmark Ed.)22, 1493–1522 (2017). - PMC - PubMed
    1. Maiti, A. & Drohat, A. C. 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 (2011). - PMC - PubMed
    1. Maiti, A., Michelson, A. Z., Armwood, C. J., Lee, J. K. & Drohat, A. C. Divergent mechanisms for enzymatic excision of 5-formylcytosine and 5-carboxylcytosine from DNA. J. Am. Chem. Soc.135, 15813–15822 (2013). - PMC - PubMed
    1. Pidugu, L. S., Dai, Q., Malik, S. S., Pozharski, E. & Drohat, A. C. Excision of 5-carboxylcytosine by thymine DNA glycosylase. J. Am. Chem. Soc.141, 18851–18861 (2019). - PMC - PubMed
    1. Pidugu, L. S. et al. Structural basis for excision of 5-formylcytosine by thymine DNA glycosylase. Biochemistry55, 6205–6208 (2016). - PMC - PubMed

LinkOut - more resources