When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions

Abstract

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3'-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide ("long-patch") BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5'-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15-30min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60-70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5'-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.

Keywords: 2-deoxyribonolactone; AP lyase; Base excision DNA repair; DNA polymerase β; DNA-protein crosslinks; Oxidative DNA damage.

Figure 1. BER processing of oxidative DNA damage and the formation of DPC

Following base damage and glycosylase processing, or the oxidation of DNA deoxyribose, the resulting abasic sites are cleaved by Ape1 (1A, B). The incised AP sites are then channeled through single-nucleotide (“short-patch”) or multinucleotide (“long-patch”) BER. In the former, following incision of the AP site, Polβ inserts a single-nucleotide to replace the damaged nucleotide (2A) and removes the 5’-dRp residue generated by Ape1 (3A). Synthesis of ≥2 nucleotides in multinucleotide BER generates a flap that prevents 5’-dRp excision and requires activities such as Fen1 (3B). Multinucleotide BER processes dL lesions effectively (2B), but in some circumstances attempted 5’-dL excision results in the formation of Polβ-DPC (2C). Short-patch BER is completed by DNA Ligase IIIα (4A) and in the case of long-patch BER, repair is completed by DNA Ligase I (4B) restoring the DNA backbone to its native condition.

Figure 2. The Fate and Impact of Oxidative Polβ-DPC in vivo

Exposure of cells to Cu(OP)₂ results in robust Polβ-DPC formation (1). Polβ-DPC are subsequently targeted for ubiquitylation (2) and rapid proteolysis, which is expected to generate 5’-peptidyl-dL-DNA adducts (3A) that would be removed from the genome by DNA repair mechanisms being investigated (4A). Together, such repair processes would allow cells to escape the toxic effects of oxidative Polβ-DPC accumulation (5A). However, inhibition of the proteasome prevents removal of Polβ-DPC (3B), leading to their toxic accumulation (4B) with cell killing and perhaps other consequences (5B). Biological and mechanistic questions requiring further definition are highlighted in brown.

Figure 3. Representative Structures of Some Mechanism-based DPC

Schematic depiction of the bonding chemistry of DPC due to DNA lesions or enzyme-inhibiting drugs (see main text). The structures are: Polβ-dL-DPC (A), hOGG1-Oxa-DPC (B), Fpg-cHyd-DPC (C), Top2-DPC (D), and Top1-DPC (E). The linking amino acid residues for Polβ, hOGG1 and Fpg are indicated using single-letter code (K = lysine; P = proline).