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.

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