Proteomics Study of DNA–Protein Crosslinks in Methylmethanesulfonate and Fe²⁺-EDTA-Exposed Human Cells

Abstract

The formation of covalently bound DNA−protein crosslinks (DPCs) is linked to the pathophysiology of cancers and many other degenerative diseases. Knowledge of the proteins that were frequently involved in forming DPCs will improve our understanding of the etiological mechanism of diseases and facilitate the establishment of preventive measures and treatment methods. By using SDS-PAGE and nano-LC coupled Orbitrap LC-MS/MS analyses, we identified, for the first time, that the major DNA-cross-linked proteins in HeLa cells exposed to a methylating agent (methylmethanesulfonate) or hydroxyl free radicals are transcription-associated proteins. In particular, histone H2B3B and poly(rC) binding protein 2 were identified as the most frequent DPC-forming proteins.

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.…”

“…The list of cross-linked sites includes multiple DNA bases (G, C, T, A) and their modifications (e.g., N 7 -Me G (12), abasic sites (13), and 5-formyl dC (10,11)). The list of proteins in the cross-links includes histones (10,12), HMCES (13), and numerous other proteins (21)(22)(23)(24)(25). Some reversible lysine cross-links (Schiff bases) can also destabilize DNA and cause cleavage (12,26).…”

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

“…In the case of the N 2 -dG-36-mer peptide cross-link, the extension efficiency was reduced as compared to the unmodified as well as the N 2 -dG-15-mer peptide cross-link template (Fig. 4B, lanes [13][14][15][16][17][18].…”

“…DNAprotein cross-links can be formed in many different ways ( 6): (i) by endogenous agents either in enzymatic (e.g., topoisomerase) (7) or non-enzymatic processes (e.g., abasic sites, formaldehyde) (8,9), (ii) by chemotherapeutic and other exogenous chemical agents (e.g., cisplatin-induced DNA-protein cross-links) ( 10), (iii) by exogenous physical damage (e.g., ionizing radiation) (11), or (iv) if a protein strongly binds to DNA and behaves as a DNAprotein cross-link. Proteins involved in crosslinking include DNA polymerase (pol) β (12), poly(ADP) ribose polymerase 1 (PARP1) (13), histones (14), DNA glycosylase (15), HMCES (16), and at least 70 others (17). DNA-protein cross-links can form at various sites in DNA, including all four natural nucleobases (dA, dC, dG, and dT).…”

“…, ionizing radiation) ( 11 ); or (iv) if a protein strongly binds to DNA and behaves as a DNA–protein cross-link. Proteins involved in cross-linking include DNA polymerase (pol) β ( 12 ), poly(ADP) ribose polymerase 1 (PARP1) ( 13 ), histones ( 14 ), DNA glycosylase ( 15 ), HMCES ( 16 ), and at least 70 others ( 17 ). DNA–protein cross-links can form at various sites in DNA, including all four natural nucleobases (dA, dC, dG, and dT).…”