Dependence of DNA–Protein Cross-Linking via Guanine Oxidation upon Local DNA Sequence As Studied by Restriction Endonuclease Inhibition

Abstract

Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones

“…Also, the MBR and MTC have a higher GC content (61% and 55%, respectively) than the genome average of S. cerevisiae (38%) and H. sapiens (41%). GC-rich sequences appear especially prone to ROS due to the oxidation of guanine (Madison et al, 2012; Saito et al, 1995) and damage from Cu—H 2 O 2 complexes (Rodriguez et al, 1995). Thus, long-lived oxidized DNA lesions may accumulate at the fragile zones and may explain the large increase in the ZBE rate we observe when ARM37 is expressed in a tsa1Δ mutant.…”

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

“…This may be rationalized in terms of the initial generation of guanine radical cations since 6-thioguanine has been shown to act as a type I photosensitizer. Further support is gained by previous model studies that have involved several one-electron oxidants (Nguyen et al 2000, Kurbanyan et al 2003, Madison et al 2012) including high intensity UVC laser irradiation (Angelov et al 2003) as generators of DPC. A detailed mechanistic study describing the covalent attachment of TGT trinucleotide to trilysine peptide (KKK) is now available (Perrier et al 2006).…”

Guanine Holes Are Prominent Targets for Mutation in Cancer and Inherited Disease

“…In the first step, abstraction of an electron from DNA (base or sugar) by a radical species, either endogenous or exogenous, creates an electron hole. In the second step, the electron hole migrates reversibly to various competing sites, including flanking or more distant bases, as well as other molecules and contacting chromatin-associated amino acids, causing in some instances DNA-protein crosslinks [64]. Guanines with the lowest ionization potentials, as determined by neighboring bases, are the strongest hole-attracting sites.…”