Biologically relevant oxidants cause bound proteins to readily oxidatively cross-link at Guanine.
Oxidative DNA-protein cross-links 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 cross-linking to occur in the presence of oxidants. Cross-link formation was examined using four different oxidation systems that generate singlet oxygen, superoxide, and metal-based Fenton reactions. It is shown that oxidative cross-linking 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 cross-links, it was chosen for further analyses. Cross-linking occurred with single-stranded DNA binding protein and not with bovine serum albumin. Addition of salt lowered nonspecific binding affinity and lowered cross-link yield by up to 59%. The yield of cross-linking increased with increased ratios of protein compared with DNA. Cross-linking 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 cross-linking, while primer extension studies showed cross-links 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, which were cross-linked to 2'-deoxyguanosine. The cross-link products were spiroiminodihydantoin, guanidinohydantoin, and tyrosyl-based adducts. Formation of tyrosine-based adducts may be competitive with the more well-studied lysine-based cross-links. We conclude that oxidative cross-links may be present at high levels in cells since the propensity to oxidatively cross-link is high and so much of the genomic DNA is coated with protein.
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, United States.
Morwena J
MJ
Solivio
Dessalegn B
DB
Nemera
Larry
L
Sallans
Edward J
EJ
Merino
D003300:Copper / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D016877:Oxidants / Q000737:chemistry*; D010084:Oxidation-Reduction; D012256:Riboflavin / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012395:Rose Bengal / Q000737:chemistry
D003432:Cross-Linking Reagents; D016877:Oxidants; D012395:Rose Bengal; D006147:Guanine; D003300:Copper; D004247:DNA; D007501:Iron; D012259:Ribonuclease, Pancreatic; D012256:Riboflavin