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

摘要

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 similar to 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 beta (Pol beta) 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 Pol beta cross-linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Pol beta and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Pol beta-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 Pol beta-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15-30 min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Pol beta-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60-70% of the trapped ubiquitin depends on Pol beta. The mechanism of ubiquitin targeting to Pol beta-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. (C) 2016 Elsevier B.V. All rights reserved.