Reconsidering the Chemical Nature of Strand Breaks Derived from Abasic Sites in Cellular DNA: Evidence for 3′-Glutathionylation

Abstract

The hydrolytic loss of coding bases from cellular DNA is a common and unavoidable reaction. The resulting abasic sites can undergo β-elimination of the 3′-phosphoryl group to generate a strand break with an electrophilic α,β-unsaturated aldehyde residue on the 3′-terminus. The work reported here provides evidence that the thiol residue of the cellular tripeptide glutathione rapidly adds to the alkenal group on the 3′-terminus of an AP-derived strand break. The resulting glutathionylated adduct is the only major cleavage product observed when β-elimination occurs at an AP site in the presence of glutathione. Formation of the glutathionylated cleavage product is reversible, but in the presence of physiological concentrations of glutathione, the adduct persists for days. Biochemical experiments provided evidence that the 3′-phosphodiesterase activity of the enzyme apurinic/ apyrimidinic endonuclease (APE1) can remove the glutathionylated sugar remnant from an AP-derived strand break to generate the 3′OH residue required for repair via base excision or single-strand break repair pathways. The results suggest that a previously unrecognized 3′glutathionylated sugar remnantand not the canonical α,β-unsaturated aldehyde end groupmay be the true strand cleavage product arising from β-elimination at an abasic site in cellular DNA. This work introduces the 3′glutathionylated cleavage product as the major blocking group that must be trimmed to enable repair of abasic site-derived strand breaks by the base excision repair or single-strand break repair pathways.

DNA–protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM)

“…Notably, Yang and colleagues also showed that the half-life of Schiff base DPCs derived from histone and 3′-pUA ranges from 10 to 14 h in vitro ( 49 ). Overall, our observations of the important role of GSH in forming GSH-adducted SSB and in competing with TFAM in the DPC formation corroborate results from the Gates and Wang laboratories showing the glutathionylation of β-elimination products of AP sites in nDNA ( 30 , 50 ). Additional studies are needed to clarify which type(s) of TFAM-induced DNA modifications are more persistent in vivo .…”

“…We verified the formation of a reactive 3′-phospho-α,β-unsaturated aldehyde residue (3′pUA, shown in Figure 2A ) by comparing products with those from well-characterized reactions from NaOH or DNA glycosylase-catalyzed reactions ( 28 ), or by trapping with thiol-containing chemicals, such as β-mercaptoethanol (βMe) or GSH. Small molecule thiols ( 29 , 30 ) or Cys residues ( 31 ) are known to be able to conjugate with an α,β-unsaturated aldehyde via 1,4-addition. As shown in Figure 2B (lanes 2, 3, 14, and 15), reactions with AP(5′) under mild and strong NaOH treatment proceeded primarily via β-elimination and β,δ-elimination, respectively.…”

Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing

“…Recently, the Gates and Guengerich laboratories have independently described thiol adducts formed at abasic sites and abasic site-derived cleavage products. − In Figure , we present a structure consistent with the observed mass, although other structural isomers are possible. Interestingly, we find that the PUA is reactive with a broad range of nucleophiles.…”

N-Methyl-N-nitrosourea Induced 3′-Glutathionylated DNA-Cleavage Products in Mammalian Cells

“…Mammalian AP-endonuclease APE1 was shown to be capable of removing the 3′-PUA generated from the β-elimination of the AP site. , APE1 may also trim GS-ddR to produce a 3′-hydroxyl terminus for polymerase β-mediated gap filling, and the endonuclease activity of APE1 on AP sites precludes the generation of GS-ddR. We used the APE1 inhibitor CRT0044867 to examine the influence of APE1 inhibition on the generation of GS-ddR.…”

“…3′-PUA exhibits high reactivity with thiol, and we recently reported the formation of the 3′-glutathionylated DNA strand-cleavage product, that is, 3′-glutathionyl-2,3-dideoxyribose (GS-ddR), in synthetic duplex DNA in vitro (Scheme ). Considering that the cellular concentration of GSH ranges from 0.5 to 10 mM, , we reason that the same product may form in cellular DNA. Here, we developed a stable-isotope-dilution-based liquid chromatography tandem mass spectroscopy (LC–MS/MS) method for the quantification of the basal level and MNU-induced formation of GS-ddR in cultured human cells.…”