5',8-Cyclopurine Lesions in DNA Damage: Chemical, Analytical, Biological, and Diagnostic Significance

Abstract

Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5'R and 5'S, for each 2'-deoxyadenosine and 2'-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2'-deoxyribose units generating C5' radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.

Keywords: DNA and RNA polymerases; DNA damage; LC-MS/MS; cancer; cyclopurines; free radicals; nucleotide excision repair; reactive oxygen species; xeroderma pigmentosum.

Figure 1

Purine 2′-deoxynucleotide reacts with a hydroxyl radical (HO^•), yielding the purine 5′,8-cyclo-2′-deoxynucleotide via cyclization of the C5′ radical followed by oxidation.

Figure 2

Libraries of purine 5′,8-cyclo-2′-deoxynucleosides (cPu).

Figure 3

Bioinspired radical transformations for the synthesis of 5′,8-cyclopurines.

Figure 4

Synthesis of (A) 5′,8-cyclo-2′-deoxyadenosine (cdA) and (B) 5′,8-cyclo-2′-deoxyguanosine (cdG) in both diastereoisomeric forms, differentiating the two secondary 3′OH and 5′OH groups, by the radical cascade protocol. (m.p.: medium pressure).

Figure 5

Comparison of the total yields of the 17-mer: 5′-d(CCA CCA ACX CTA CCA CC)-3′, where X = S-cdA, R-cdA, S-cdG, or R-cdG.

Figure 6

(A) Schematic illustrations of the placements of the DNA lesions and the Cy3 donor and Cy5 acceptor molecules at the “Out” and “In” rotational settings in the 147-mer 601 DNA duplexes used in the Förster Resonance Energy Transfer (FRET) experiments. (B) The crystal structure of the 601 nucleosome core particles (PDB 3LZ0) [67]. The dyad axis is indicated by the red line. (C) Positions of the Cy3 donor and Cy5 acceptor molecules in the nucleosome FRET experiments. The lesions were positioned at the 66th or 70th nucleotide (nt) counted from the 5′-end of the 147-mer, corresponding to the Out or In rotational settings. The internal Cy3 and Cy5 labels were positioned at nucleotides 43 and 39 counted from the dyad axis in opposite strands. Reproduced from reference [66].

Figure 7

Representative autoradiographs of denaturing gels of the results of nucleotide excision experiment assays in HeLa cell extracts. (A) The substrates were either free 147-mer 601^cP DNA sequences containing single cPu lesions or nucleosomes assembled with histone octamers derived from recombinant (Rec) histones or native, post-translationally modified histones extracted from HeLa cells. (B) Analogous nucleotide excision repair (NER) experiments with cis- and trans-B[a]PDE-dG adducts positioned at the same In and Out superhelical locations. Three separate gels are depicted in panels A and B. Reproduced from reference [66].

Figure 8

Accumulation of cPu disrupts DNA replication, repair, and gene transcription, leading to lesion bypass, mutations, and genome instability.

Figure 9

A cdA located at DNA repeat sequences induces repeat instability through pol β bypass of a loop structure. Pol β: DNA polymerase β; LIG I: DNA ligase I; FEN1: flap endonuclease 1.

Figure 10

(A) MS/MS fragmentation spectra (ESI–MS/MS of the [M + H]⁺ ion) of S-cdA (m/z 250→164) and S-cdG (m/z 266→180) lesions. Similar fragment ions were observed for R-cdA and R-cdG lesions. (B) MS/MS fragmentation spectra (ESI–MS/MS of the [M + H]⁺ ion) of isotopically labeled (N = ¹⁵N) S-cdA (m/z 255→169) and S-cdG (m/z 271→185) lesions.

Figure 11

Workflow showing protocol steps for the quantification of cPu lesions via isotope dilution LC–ESI–MS/MS (the fragmentation pattern of the lesions is shown); dC: 2′-deoxycytidine; dG: 2′-deoxyguanosine; Thy: thymidine; dA: 2′-deoxyadenosine.

Figure 12

Levels of cPu lesions/10⁶ nucleosides measured by LC–MS/MS from breast cancer MDA-MB-231 and MCF-7 cells with or without exposure to γ-irradiation (upper part) and hydrogen peroxide (lower part). The light bars (blue and green, respectively) represent samples exposed to 5 Gy or 300 μΜ H₂O₂ followed by a 1 h repair period, and the dark bars (blue and green, respectively) represent samples exposed to 5 Gy or 300 μΜ H₂O₂ followed by a 24 h repair period. The white bars represent untreated samples (control). The asterisks denote a statistically significant difference (p < 0.05) between the untreated controls and the treated samples. From reference [133].

Figure 13

Levels of cPu lesions/10⁶ nucleosides measured by LC–MS/MS in control severe combined immunodeficient (SCID) and tumor-bearing SCID mice. (A) Levels of R-cdG, S-cdG, R-cdA, and S-cdA lesions in genomic DNA isolated from the liver of control SCID mice (group 1: 4 weeks old; group 2: 17 weeks old) and tumor-bearing SCID mice (group 3: 4 weeks old; group 4: 17 weeks old). (B) Levels of R-cdG, S-cdG, R-cdA, and S-cdA lesions in genomic DNA isolated from the kidney of control SCID mice (groups 1 and 2) and tumor-bearing SCID mice (groups 3 and 4). * Denotes a statistically significant difference (p < 0.05) and ** denotes a statistically significant difference p < 0.005 between the animal groups. From reference [140].