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Proteome-Wide Identification of O6-Methyl-2'-deoxyguanosine-Binding Proteins
Andrew H Kellum1, Michelle Y Wang1,2, Ting Zhao3
1Department of Chemistry, University of California Riverside, Riverside, California 92521-0403, United States.
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.
Insights
Researchers identified proteins that recognize O6-methyl-2'-deoxyguanosine (O6-Me-dG) DNA damage. Loss of CDKN2AIP (CARF) protein increased sensitivity to DNA methylating agents, revealing its role in DNA damage response.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA alkylation damage, particularly O6-methyl-2 '-deoxyguanosine (O6-Me-dG), poses a mutagenic threat.
- While repair pathways for O6-Me-dG exist, the cellular proteins that recognize this lesion remain largely uncharacterized.
Purpose of the Study:
- To identify novel DNA reader proteins that specifically recognize O6-Me-dG lesions.
- To investigate the functional role of identified reader proteins in cellular response to DNA alkylation damage.
Main Methods:
- Quantitative mass spectrometry was employed to identify proteins binding to O6-Me-dG in DNA duplexes.
- Genetic ablation of candidate proteins (CDKN2AIP/CARF) in HEK293T cells.
- Assessment of cellular tolerance to N-nitroso-N-methylurea (NMU) and quantification of genomic O6-Me-dG levels.
Main Results:
- 67 and 31 candidate reader proteins were identified for O6-Me-dG:2 '-deoxycytidine and O6-Me-dG:thymidine base pairs, respectively.
- CDKN2AIP (CARF) was identified as a protein recognizing both O6-Me-dG:dC and O6-Me-dG:dT base pairs.
- Genetic ablation of CDKN2AIP led to increased sensitivity to NMU and reduced accumulation of O6-Me-dG in genomic DNA.
Conclusions:
- This study reveals novel reader proteins for the mutagenic O6-Me-dG DNA lesion.
- CDKN2AIP (CARF) plays an unexpected role in sensitizing cells to DNA methylating agents, highlighting its involvement in DNA damage response pathways.
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