Synthesis and Excision Repair of Site-Specific 3′-End DNA-Histone Cross-Links Derived from Abasic Sites

Abstract

Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA−protein cross-links (DPCs) at 3′-DNA termini within single-strand breaks. These DPCs need to be removed because 3′-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3′-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3′-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 sitespecifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.

Mass Spectrometry Evidence for Forming Schiff Base 3′-DNA-Histone Cross-Links from Abasic Sites in Vitro and in Human Cells

“…The traditional sample preparation involves a sequential DPC digestion by a protease (e.g., trypsin) and a nuclease cocktail . A limitation is that the steric hindrance imposed by the cross-linked peptide can inhibit DNA digestion, resulting in a heterogeneous mixture of modified peptides that are not suitable for mass spectrometry analysis. , We recently developed an LC-MS/MS workflow to characterize 3′-DPCs formed through Michael addition by using a 3′-DPC repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1), as a molecular tool to specifically release tryptic 3′-DPCs to yield homogeneous samples. − In this study, we developed a similar LC-MS/MS pipeline to detect Schiff base 3′-histone-DPCs. Our method (Figure B) involves four major steps: (1) stabilizing 3′-histone-DPCs by NaBH 4 reduction, (2) excising intact histones by excess TDP1, (3) acylating modified histones by propionic anhydride before and after trypsin digestion to improve the separation of histone peptides on HPLC, and (4) LC-MS/MS analysis and identification of histone peptides that contain a dynamic modification of K + 100.0524 Da.…”

“…Unrepaired 3′-histone-DPCs are extremely toxic, as they will block DNA replication and transcription. We recently demonstrated that 3′-histone-DPCs can be efficiently repaired by three nucleases including TDP1, AP endonuclease 1, and three-prime repair exonuclease 1, but only when the cross-linked histones are initially digested by trypsin . How 3′-histone-DPCs are degraded in cells warrants future investigation.…”

Dual-responsive probe and DNA interstrand crosslink precursor target the unique redox status of cancer cells

“…We determined the alkylation sites based on the fact that alkylated adducts were usually heat susceptible under alkaline conditions, leading to strand breaks at alkylation sites. 29,30 It was found that A5, G6, A14, A15, A18, G22, C23, A24, A25, and G27 were the major cleavage sites. Meanwhile cleavage was also found at C3, C9, and C19 for 15 treated with 14 (Fig.…”