Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form unstable but long-lived Schiff base DNA–protein cross-links at 3′-DNA termini (3′-PUA–protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1’s intrinsic redox capacity, yielding a stable 3′-PUA–PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3′-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3′-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3′-PUA–protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3′-PUA–histone DPCs, but not 3′-PUA–PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3′-PUA–protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Detailed kinetic analysis reveals a complex multi-step mechanism for an amine-thiol “declick” reaction.

Silica passivating agents have shown great success in minimizing nonspecific protein binding to glass surfaces for imaging and microscopy applications. Amine-derivatized surfaces are commonly used in conjugation with amide coupling agents to immobilize peptides/proteins through C-terminal or side-chain carboxylic acids. In the case of the single-molecule fluorosequencing of peptides, attachment occurs via the C-terminus and nonspecific surface binding has previously been a source of error in peptide identification. Here, we employ fluorosequencing as a high-throughput, single-molecule sensitivity assay to identify and quantify the extent of nonspecific binding of peptides to amine-derivatized surfaces. We show that there is little improvement when using common passivating agents in combination with the surface derivatizing agent 3-amino­propyl-tri­ethoxy­silane (APTES) to couple the peptides to the modified surface. Furthermore, many xanthene fluorophores have carboxylic acids in the appended phenyl ring at positions ortho and meta or ortho and para, and the literature shows that conjugation through the ortho position is not favored. Because xanthene-derived fluorophores are commonly used for single-molecule applications, we devised a novel assay to probe the conjugation of peptides via their fluorophores relative to their C-termini on silane-derivatized surfaces. We find significant attachment to the ortho position, which is a warning to those attempting to immobilize fluorophore-labeled peptides to silica surfaces via amide coupling agents. However, eliminating all amines on the surface by switching to 3-azido­propyl-tri­ethoxy­silane (AzTES) for coupling via copper-catalyzed azide–alkyne cycloaddition (CuAAC) and omitting additional passivation agents allowed us to achieve a high level of C-terminally bound peptides relative to nonspecifically or ortho-phenyl-bound, fluorophore-labeled peptides. This strategy substantially improves the specificity of peptide immobilization for single-molecule fluorosequencing experiments.