Biologically Relevant Oxidants Cause Bound Proteins To Readily Oxidatively Cross-Link at Guanine

Abstract

Oxidative DNA-protein crosslinks have received less attention than other types of DNA damage and remain as one of the least understood types of oxidative lesion. A model system using Ribonuclease A and a 27-nucleotide DNA was used to determine the propensity of oxidative crosslinking to occur in the presence of oxidants. Crosslink formation was examined using four different oxidation systems that generate singlet oxygen, superoxide, and metal-based Fenton reactions. It is shown that oxidative crosslinking occurs in yields ranging from 14% to a maximal yield of 61% in all oxidative systems when equivalent concentrations of DNA and protein are present. Because singlet oxygen is the most efficient oxidation system in generating DNA-protein crosslinks, it was chosen for further analyses. Crosslinking occurred with single-stranded DNA binding protein and not with bovine serum albumin. Addition of salt lowers non-specific binding affinity and lowered crosslink yield by up to 59%. The yield of crosslinking increased with increased ratios of protein compared to DNA. Crosslinking was highly dependent on the number of guanines in a DNA sequence. Loss of guanine content on the 27-nucleotide DNA led to nearly complete loss in crosslinking, while primer extension studies showed crosslinks to predominantly occur at guanine base on a 100-nucleotide DNA. The chemical species generated were examined using two peptides derived from the Ribonuclease A sequence: N-acetyl-AAAKF and N-acetyl-AYKTT that were crosslinked to 2’-deoxyguanosine. The crosslink products were spiroiminodihydantoin, guanidinohydantoin, and tyrosyl-based crosslink adducts. Formation of tyrosine based adducts may be competitive with the more well-studied lysine-based crosslinks. We conclude that oxidative crosslinks may be present at high levels in cells since the propensity to oxidatively crosslink is high and so much of the genomic DNA is coated with protein.

Ruthenium dihydroxybipyridine complexes are tumor activated prodrugs due to low pH and blue light induced ligand release

“…In addition, most Ru 2+ and Pt 2+ complexes are cytotoxic when a ligand is lost to form the di-aqua complex. This di-aqua complex can exchange the water ligands with biomolecules to induce toxicity [4, 50]. Significantly important within these ligand exchange reactions is the addition of the nucleobase guanine at the N7-positon.…”

PNA–Rose Bengal Conjugates as Efficient DNA Photomodulators

“…In turn, spirominodihydantoin and guanidinohydantoin adducts of 8-oxo-G readily react with nucleophiles such as amines of L-lysine that are found in peptides and proteins. 38,39 Singlet oxygen was found to have an important role in the mechanism of photoreaction by PNA-RB conjugates. Repeating the irradiation experiment with K-PEG-K-G-RB in D 2 O resulted in higher DNA photomodulation (Figure 4).…”

Excessive Reactive Oxygen Species and Exotic DNA Lesions as an Exploitable Liability

“…90 Formation of hydantoin–protein cross-links in the presence of DNA binding proteins at biologically relevant oxidant conditions suggests DPCs are a major type of oxidative lesions. 57 DPCs are observed at levels of 0.5–4.5 per 10 7 bases in human white blood cells. 91 Recently, the Greenburg group reported proteinlike radical formation in nucleosome core particles under oxidative stress conditions that initially shields DNA from oxidation; ultimately, DNA is damaged in an oxygen-dependent manner, showing the interplay of DNA and bound proteins in effects of oxidative stress.…”