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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
Published on: June 19, 2018
Tyrosyl-DNA phosphodiesterase 1 excises the 3'-DNA-ALKBH1 cross-link and its application for 3'-DNA-ALKBH1 cross-link
Xiaoying Wei1, Maria D Person2, Kun Yang3
1Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, United States; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, United States.
Abstract:
The apurinic/apyrimidinic (abasic, AP) site is one of the most abundant DNA lesions. Previous studies by others demonstrated that human AlkB homologue 1 (ALKBH1) catalyzes the DNA strand incision at an AP site, resulting in suicidal cross-linking of the enzyme to the 3'-DNA end. Prior site-directed mutagenesis experiments had reported that Cys129 of ALKBH1 is the predominant nucleophile that conjugates to the C3' position of the incised AP site, 3'-phospho-α,β-unsaturated aldehyde (3'-PUA), to form a 3'-PUA-ALKBH1 cross-link. However, direct evidence to support this mechanism was lacking. The 3'-PUA-ALKBH1 cross-link is so far the only adduct that has been found to form via a Michael addition reaction between a protein and 3'-PUA. It is unclear whether and how this type of cross-link is repaired. In this study, we first demonstrated that the 3'-PUA-ALKBH1 cross-link is fairly stable under physiological temperature and pH as only ~10% of the adduct decomposed after a 3-day incubation. Using a gel-based assay with an aldehyde-reacting probe, we demonstrated that the 3'-PUA-ALKBH1 cross-link has a free aldehyde group that is in line with the Michael addition mechanism. Moreover, we found that the 3'-PUA-ALKBH1 cross-link can be excised by human tyrosyl-DNA phosphodiesterase 1 (TDP1) and the removal efficiency is significantly enhanced if the adduct is pre-digested by trypsin. Notably, we employed TDP1 as a molecular tool to homogeneously release the cross-linked peptides from DNA to facilitate liquid chromatography tandem mass spectrometry analysis, and demonstrated that Cys129 and Cys371 of ALKBH1 cross-link to 3'-PUA.
Insights
Human AlkB homologue 1 (ALKBH1) forms a stable cross-link with DNA lesions. Tyrosyl-DNA phosphodiesterase 1 (TDP1) can repair this adduct, identifying Cys129 and Cys371 as cross-linking sites.
Area of Science:
- Biochemistry
- Molecular Biology
- DNA Repair
Background:
- Apurinic/apyrimidinic (AP) sites are common DNA lesions.
- Human AlkB homologue 1 (ALKBH1) incises DNA at AP sites, forming a 3'-phospho-α,β-unsaturated aldehyde (3'-PUA) and cross-linking to the DNA.
- Cys129 was proposed as the nucleophile forming the 3'-PUA-ALKBH1 cross-link via Michael addition, but direct evidence was lacking.
Purpose of the Study:
- To provide direct evidence for the ALKBH1-3'-PUA cross-linking mechanism.
- To investigate the stability and repair of the 3'-PUA-ALKBH1 cross-link.
- To identify the specific cysteine residues involved in the cross-linking.
Main Methods:
- Incubation of the 3'-PUA-ALKBH1 cross-link under physiological conditions.
- Gel-based assay with an aldehyde-reacting probe.
- Enzymatic digestion with human tyrosyl-DNA phosphodiesterase 1 (TDP1) and trypsin.
- Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
Main Results:
- The 3'-PUA-ALKBH1 cross-link is stable at physiological temperature and pH.
- The cross-link possesses a free aldehyde group, consistent with Michael addition.
- Human TDP1 can excise the 3'-PUA-ALKBH1 cross-link, with enhanced efficiency after trypsin pre-digestion.
- LC-MS/MS analysis confirmed that Cys129 and Cys371 of ALKBH1 cross-link to 3'-PUA.
Conclusions:
- Direct evidence supports the Michael addition mechanism for 3'-PUA-ALKBH1 cross-link formation involving Cys129 and Cys371.
- The 3'-PUA-ALKBH1 cross-link is a stable DNA adduct.
- Human TDP1 is capable of repairing this specific DNA-protein cross-link.
