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. 2025 Jul 29;97(29):16011-16018.
doi: 10.1021/acs.analchem.5c03119. Epub 2025 Jul 17.

Proteome-Wide Identification of O6-Methyl-2'-deoxyguanosine-Binding Proteins

Andrew H Kellum Jr  1 , Michelle Y Wang  1   2 , Ting Zhao  3 , Xiaochuan Liu  1 , Xiaomei He  1 , Lin Li  1 , Preston Williams  1 , Quanqing Zhang  1 , Yinsheng Wang  1
Affiliations

Proteome-Wide Identification of O6-Methyl-2'-deoxyguanosine-Binding Proteins

Andrew H Kellum Jr et al. Anal Chem. .

Abstract

DNA is subjected to damage from various endogenous and exogenous sources of alkylating agents, resulting in alkylated DNA lesions. Among these lesions, O6-methyl-2'-deoxyguanosine (O6-Me-dG) is highly mutagenic, and it can be repaired by O6-alkylguanine DNA alkyltransferase and mismatch repair pathway. It, however, remains unclear whether O6-Me-dG in DNA can be recognized by other cellular proteins. Here, we employed a quantitative mass spectrometry-based approach to uncover reader proteins of O6-Me-dG in DNA when it is paired with a 2'-deoxycytidine (dC) or thymidine (dT). We were able to identify 67 and 31 candidate reader proteins for duplex DNA harboring O6-Me-dG:dC and O6-Me-dG:dT base pairs, respectively. In addition, genetic ablation of CDKN2AIP, a.k.a. CARF, one of those proteins that can recognize both the O6-Me-dG:dC and O6-Me-dG:dT base pairs, in HEK293T cells conferred augmented tolerance to N-nitroso-N-methylurea (NMU), an alkylating agent that can induce O6-Me-dG in DNA. Accordingly, our LC-MS/MS quantification results revealed that the loss of CDKN2AIP led to diminished accumulation of NMU-induced O6-Me-dG in genomic DNA. Together, we explored the damage recognition proteins of O6-Me-dG using a quantitative mass spectrometry-based approach, and our results revealed an unexpected role of CDKN2AIP in sensitizing cultured cells toward a DNA methylating agent.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Base pairing configurations of O6-Me-dG when paired with dC (wobble pair) or dT (pseudo-Watson–Crick pair) (a) and forward SILAC-based interaction screening for identifying novel damage recognition proteins of O6-Me-dG (b). The “B” in red circle indicates 5′-biotin labeling.
Figure 2.
Figure 2.
SILAC-based interaction screening led to the identification of candidate recognition proteins for duplex DNA containing an O6-Me-dG:dC or O6-Me-dG:dT base pair. Scatter plots showing the proteins identified from forward and reverse SILAC pull-down assays using an O6-Me-dG:dT DNA probe relative to a dA:dT probe with nuclear protein lysates (a), O6-Me-dG:dC DNA probe relative to the dG:dC probe with nuclear protein lysates (b), and a Venn diagram displaying the overlap in interacting proteins of O6-Me-dG:dC and O6-Me-dG:dT (c).
Figure 3.
Figure 3.
ESI-MS ((a, c) forward SILAC, (b, d) reverse SILAC) showing the [M + 2H]2+ ions of the light and heavy lysine-containing peptide, LTSLNEEYTK derived from MSH2 in O6-Me-dG:dT versus dA:dT pulldown (a, b) and O6-Me-dG:dC versus dG:dC pulldown (c, d). The calculated m/z for the monoisotopic peak of the [M + 2H]2+ ion of the light lysine-containing peptide is 599.3035.
Figure 4.
Figure 4.
ESI-MS showing the [M+2H]2+ ions of light and heavy lysine-containing peptides, PSSETASSGLTSK from CDKN2AIP in the O`-Me-dG:dT versus dA:dT pulldown (a, b), and light and heavy lysine-containing peptide GISSSNEGVEEPSK from CDKN2AIP in the O6-Me-dG:dC versus dG:dC pulldown (c, d). The calculated m/z values for the monoisotopic peaks of the [M + 2H]2+ ions of the light lysine-containing peptides of PSSETASSGLTSK and GISSSNEGVEEPSK are m/z 626.3068 and 710.3335, respectively.
Figure 5.
Figure 5.
Loss of CDKN2AIP rendered HEK293T cells more resistant to NMU and conferred attenuated accumulation of NMU-elicited O6-Me-dG in genomic DNA. (a) Clonogenic survival assay results for HEK293T and the isogenic CDKN2AIP−/− cells upon exposure to different concentrations of NMU (n = 3). (b) LC-MS/MS quantification results of O6-Me-dG in the genomic DNA of HEK293T and the isogenic CDKN2AIP−/− cells harvested immediately and at 22 h after a 1.0-h exposure to 200 μM NMU. The p values were calculated by using two-way ANOVA with Sidak’s multiple comparisons test: *p < 0.05; ns, p > 0.05.
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References

    1. Rydberg B; Lindahl T EMBO J. 1982, 1, 211–216. - PMC - PubMed
    1. Mirvish SS Cancer Lett. 1995, 93, 17–48. - PubMed
    1. Hecht SS Chem. Res. Toxicol. 1998, 11, 559–603. - PubMed
    1. Fu D; Calvo JA; Samson LD Nat. Rev. Cancer 2012, 12, 104–120. - PMC - PubMed
    1. Shrivastav N; Li D; Essigmann JM Carcinogenesis 2010, 31, 59–70. - PMC - PubMed

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