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.

Mass Spectrometry‐Based Tools to Characterize DNA–Protein Cross‐Linking by Bis‐Electrophiles

“…Although traditional bottom‐up mass spectrometry‐based proteomics methodologies can help identify protein constituents of DPCs, they are unable to quantify DPC amounts after exposure to cross‐linking agents. Background levels of DPCs are present in all living cells due to endogenous exposure to reactive oxygen species and products of lipid peroxidation, and due to inadvertent trapping of DNA topoisomerases, polymerases and repair proteins on their DNA substrates . To distinguish between endogenous and exposure‐mediated cross‐linking, the amounts of DPCs in control and treated cells must be compared.…”

Risky repair: DNA-protein crosslinks formed by mitochondrial base excision DNA repair enzymes acting on free radical lesions

“…The 5’-dRP of Polβ attacks the oxidized carbon using a lysine nucleophile, generating a DPC anchored by an amide bond. Recently, the in vivo formation and removal of Polβ-DPC formed by oxidative agents was characterized in human and mouse cells [51,52]. Formation of Polβ DPC was a result of Polβ’s mechanistic role in BER.…”

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

“…AP sites are usually handled by the BER pathway, where an AP endonuclease, mainly APE1 in mammalian cells, incises AP sites, thus generating ssDNA breaks with 3′-OH and 5′-deoxyribose-5′-phosphate (5′-dRp) termini. Pol β, through its 5′-dRp-lyase activity residing in its 8-kDa domain, can remove the 5′-dRp residue [ 22 , 23 ]. Pol λ possesses a homologous 8-kDa domain, which also allows elimination of 5′-dRp [ 24 ].…”

“…Therefore, LP BER is activated in order to process such lesions. However, during unsuccessful attempts to repair L through SP BER, when Pol β attacks the 5′-dRp residue through the active site of its N-terminal lysine 72 (K72), it becomes covalently trapped on DNA via the formation of an amide bond with K72, resulting in the formation of DNA-protein cross-links (DPC) [ 22 , 23 , 24 , 26 , 27 ].…”