Quantification of a Novel DNA–Protein Cross-Link Product Formed by Reacting Apurinic/Apyrimidinic Sites in DNA with Cysteine Residues in Protein by Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method

Abstract

Emerging evidence suggests that cross-links formed by reacting DNA lesions with proteins may play a significant role in the pathophysiology of human cancer and degenerative diseases. The goal of this study was to develop a method involving liquid chromatography-tandem mass spectrometry (LC–MS/MS) coupled with the stable isotope-dilution method to quantify DNA–protein cross-link (DPC). A novel type of cross-link involving a S-glycosidic linkage formed by reacting an abasic site in DNA with the cysteine residues in protein was targeted in this study. The method entails hydrolysis of the cross-link to a 2′-deoxyribose-cysteine adduct, addition of isotopically labeled internal standard, and quantitation by LC–MS/MS analysis. The accuracy and precision of the method were evaluated with a synthetic peptide containing the cross-link. The validated method was then applied to quantitate the levels of the DNA–protein cross-link in vitro and in HeLa cells exposed to alkylating agent methylmethanesulfonate (MMS). The analysis detected dosage-dependent formation of the cross-link in both purified DNA (6.0 ± 0.6 DPC per 106 nt μM–1 MMS) and in human cells (7.8 ± 1.2 DPC per 106 nt mM–1 MMS). With the abasic site being one of the most common DNA lesions produced continuously by multiple pathways, the results provide significant new knowledge for better understanding the potential biological implications of its associated DNA–protein cross-link.

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

“…5B, lanes 2-13) and for the N 6 -dA-peptide template was dTTP > dATP > dCTP > dGTP (Fig. 5B, lanes [14][15][16][17][18][19][20][21][22][23][24][25]. hPol ι and κ only added dTTP across both templates (Fig.…”

“…Several laboratories have shown that both reversible and irreversible DNA-protein cross-links can be formed under physiological conditions, that there are DNA-stimulated proteases that can act on these, and that DNA-protein cross-links can be bypassed and can miscode (2,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The number of cross-links in a cell has been estimated to be high (20) but the exact number is not known.…”

In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

“…An AP site is one of the most abundant endogenous DNA lesions ( 2 ). It acts as an electrophile that can react with protein nucleophiles ( e.g., lysine and cysteine residues) to yield various types of covalent DPCs including Schiff base ( 16 , 17 , 18 , 22 , 24 , 25 , 45 , 46 ), thiazolidine ( 47 , 48 , 49 , 50 ), S -glycosidic ( 51 ), and N -glycosidic ( 52 , 53 ) bond-linked AP-protein adducts. These DPCs are either new types of DNA lesions or proposed to temporarily protect the lesions from the error-prone repair ( 17 , 49 ).…”

DNA- and DNA-Protein-Crosslink Repair in Plants

“…Interstrand crosslinking by ROSs mainly derives from C4′-oxidized abasic sites and nucleophilic addition to guanine radical cations [17]. DPCs can moreover indirectly arise from ROSs via the formation of apyrimidinic/apurinic (AP) sites, leading to covalent linkages of nearby proteins [6,18]. Apart from ICLs and DPCs, oxidative DNA damage further includes intrastrand CLs by bonding a nucleobase with the 5′ carbon of the 2-deoxyribose from the same nucleobase or a neighboring pyrimidine base [19,20].…”