Oxidation of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Leads to Substantial DNA-Histone Cross-Links within Nucleosome Core Particles

Abstract

8-Oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) is a common primary product of cellular oxidative DNA damage. 8-OxodGuo is more readily oxidized than 2’-deoxyguanosine (dG); a two-electron oxidation generates a highly reactive intermediate (OGox), which forms covalent adducts with nucleophiles, including OH−, free amines, and the side chains of amino acids such as lysine. We determined here that K3Fe(CN)6 oxidation of 8-oxodGuo in nucleosome core particles (NCPs) produces high yields, quantitative (i.e. 100%) in some cases, of DNA-protein cross-links (DPCs). The efficiency of DPC formation was closely related to 8-oxodGuo base pairing and location within the NCP and was only slightly decreased by adding the DNA-protective polyamine spermine to the system. Using NCPs that contained histone mutants, we determined that DPCs result predominantly from OGox trapping by the N-terminal histone amine. The DPCs were stable under physiological conditions and therefore could have important biological consequences. For instance, the essentially quantitative yield of DPCs at some positions within NCPs would reduce the yield of the mutagenic DNA lesions spiroiminodihydantoin and guanidinohydantoin produced from the common intermediate OGox, which in turn would affect mutation signatures of oxidative stress in a position dependent manner. In summary, our findings indicate that site-specific incorporation of 8-oxodGuo into NCPs, followed by its oxidation, leads to DPCs with an efficiency depending on 8-oxodGuo location and orientation. Given that 8-oxodGuo formation is widespread in genomic DNA and that DPC formation is highly efficient, DPCs may occur in eukaryotic cells and may affect several important biological processes.

Excision of Oxidatively Generated Guanine Lesions by Competing Base and Nucleotide Excision Repair Mechanisms in Human Cells

“…13 The low levels of the hydantoin lesions in cellular environment can be associated with the competitive formation of other oxidation products, 14 including crosslinks between 8-oxoG and histones in nucleosomes in relatively high yields. 15 Although the cellular levels of hydantoin lesions are low, they can contribute to the malignant transformation of cells because they are at least one order of magnitude more mutagenic than 8-oxoG. 16 Recent observations suggest that the hydantoin lesions are not only excellent BER substrates, [17][18][19][20][21] but are also excised by the NER pathway in human cell-free extract.…”

“…Existing paradigms suggest that nonbulky oxidatively generated DNA lesions are removed by base excision repair (BER) mechanisms, while bulky DNA double helix-distorting lesions are excised by the nucleotide excision repair (NER) pathway. − For example, DNA lesions such as oxidatively generated 8-oxo-7,8-dihydroguanine (8-oxoG) are known to be substrates of BER only. − The 8-oxoG lesions are more easily oxidized than the parent base guanine, and their oxidation can lead to the formation of stereoisomeric spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. − The accumulation of Sp lesions was detected in Nei-deficient Escherichia coli cells treated with chromate and were also detected in both the liver and colon tissues of Rag 2 –/– mice at levels ∼100 times lower than those of 8-oxoG . The low levels of the hydantoin lesions in cellular environment can be associated with the competitive formation of other oxidation products, including cross-links between 8-oxoG and histones in nucleosomes in relatively high yields . Although the cellular levels of hydantoin lesions are low, they can contribute to the malignant transformation of cells because they are at least 1 order of magnitude more mutagenic than 8-oxoG …”

Histones participate in base excision repair of 8-oxodGuo by transiently cross-linking with active repair intermediates in nucleosome core particles

“…The dsDNA sequence used in this study was 145 bp ‘601’ DNA, which forms stable, well-positioned NCPs ( 26 , 27 ). Following the procedure we reported previously ( 23 ), we prepared free dsDNA and NCPs containing a single 8-oxodGuo/C base pair at each of positions 73, 89 and 137 (Figure 2A ). To facilitate subsequent gel imaging, we labelled the 8-oxodGuo-modified strand with a 5′-FAM group.…”

“…dsDNA and NCPs containing a single 8-oxodGuo/C base pair (referred to as dsDNA-8-oxodGuo and NCP-8-oxodGuo, respectively) at three different positions (73, 89 and 137) were prepared according to the procedure we reported previously ( 23 ). dsDNA and NCPs containing a single AP at position 137 (referred to as dsDNA-AP 137 and NCP-AP 137 , respectively) were obtained by in situ photolysis of dsDNA and NCPs containing a photoprotected AP 137 ( 21 , 24 ).…”

A Photosensitized Singlet Oxygen (1O2) Toolbox for Bio-Organic Applications: Tailoring 1O2 Generation for DNA and Protein Labelling, Targeting and Biosensing

“…More specifically, the increased electrophilicity of the C5 position makes 8-oxo-dG suitable for reaction with the amino groups of proteins (e.g., lysine lateral chains), leading to the formation of irreversible protein–DNA CL adducts [ 63 ]. Using histone mutants, Bai et al demonstrated that G-oxidation can, in some cases, produce quantitative DNA–protein adducts in nucleosome-core particles [ 64 ]. Although underestimated, this type of lesion can have deleterious consequences for the normal DNA biology, as it can disrupt the normal disposition of the double helix, blocking the transcription and replication of the DNA and limiting the repair mechanisms [ 65 ].…”