RESUMEN

Free radicals produce in DNA a large variety of base and deoxyribose lesions that are corrected by the base excision DNA repair (BER) system. However, the C1'-oxidized abasic residue 2-deoxyribonolactone (dL) traps DNA repair lyases in covalent DNA-protein crosslinks (DPC), including the core BER enzyme DNA polymerase beta (Polß). Polß-DPC are rapidly processed in mammalian cells by proteasome-dependent digestion. Blocking the proteasome causes oxidative Polß-DPC to accumulate in a ubiquitylated form, and this accumulation is toxic to human cells. In the current study, we investigated the mechanism of Polß-DPC processing in cells exposed to the dL-inducing oxidant 1,10-copper-ortho-phenanthroline. Alanine substitution of either or both of two Polß C-terminal residues, lysine-206 and lysine-244, enhanced the accumulation of mutant Polß-DPC relative to the wild-type protein, and removal of the mutant DPC was diminished. Substitution of the N-terminal lysines 41, 61, and 81 did not affect Polß-DPC processing. For Polß with the C-terminal lysine substitutions, the amount of ubiquitin in the stabilized DPC was lowered by ∼40 % relative to wild-type Polß. Suppression of the HECT domain-containing E3 ubiquitin ligase TRIP12 augmented the formation of oxidative Polß-DPC and prevented Polß-DPC removal in oxidant-treated cells. Consistent with the toxicity of accumulated oxidative Polß-DPC, TRIP12 knockdown increased oxidant-mediated cytotoxicity. Thus, ubiquitylation of lysine-206 and lysine-244 by TRIP12 is necessary for digestion of Polß-DPC by the proteasome as the rapid first steps of DPC repair to prevent their cytotoxic accumulation. Understanding how DPC formed with Polß or other AP lyases are repaired in vivo is an important step in revealing how cells cope with the toxic potential of such adducts.

RESUMEN

Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polß) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polß-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polß-DPC accumulation and trapped ubiquitin in the DPC, with Polß accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polß: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polß, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polß-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

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RESUMEN

Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase ß (Polß) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polß-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polß-DPC in vivo. In a dose-dependent fashion, Polß-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polß-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polß-DPC. Nonoxidative agents did not generate Polß-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polß-DPC depended on the Ape1 AP endonuclease, which generates the Polß lyase substrate, and they required the essential lysine-72 in the Polß lyase active site. Oxidative Polß-DPC had an unexpectedly short half-life (∼ 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polß than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polß-DPC and the biological pressure to repair them.

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Mercury flux from HgCl2-treated sand and untreated soil samples of varying thickness (0.5-15 mm) were measured in dark and light under a Teflon dynamic flux chamber. Mean emissions over a 5.5-d sampling period showed an increase with depth for sand samples between 0.5 and 2 mm, but increasing depth above 2 mm had no effect. First-order kinetic models showed strong goodness of fit to the data and explained a high degree ofvariability in the emissions profile of all sand samples (R = 0.70-0.98). Soil samples showed an initial emissions peak that was not correlated with depth, suggesting a very shallow process at work. However, longer-term "baseline" emissions, measured as mean emissions between days 4.5 and 5.5, did show a relationship with depth. First-order kinetic models showed good fit for soil samples up to 4 mm thick (R2 = 0.66-0.91); however, thicker samples did not show a consistent fit to first- or second-order kinetic models (1 degree R2 = 0.00-0.46; 2 degree R2 = 0.00-0.54). The data suggest that mercury emissions from soil samples may follow a multicomponent model for which more

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