DNA-protein Crosslinks In Mammalian Cells Research Articles

Isolation and detection of DNA-protein crosslinks in mammalian cells.

DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

SPRTN patient variants cause global-genome DNA-protein crosslink repair defects

DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.

Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

SUMMARYDNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5′ terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5′ terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.

Oxidative DNA-protein crosslinks formed in mammalian cells by abasic site lyases involved in DNA repair

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 (Polβ) 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 Polβ-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polβ-DPC accumulation and trapped ubiquitin in the DPC, with Polβ 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 Polβ: 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 Polβ, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polβ-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

Abstract 4045: Decreased Fanconi anemia gene expression contributes to efficacy of PARP and DNMT inhibitor combination therapy in triple negative breast cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have efficacy in a sub-set of triple negative breast cancers (TNBCs) with inherited mutations in DNA double strand break repair (DSBR) genes, such as the BRCA1/2 genes in homologous recombination (HR), through synthetic lethality. However, PARP inhibitors have failed for the majority of sporadic TNBCs with intact BRCA1 genes. Therefore, novel targeted therapies must be explored. We have recently reported that PARPi Talazoparib in combination with DNA methyl transferase inhibitors (DNMTi)s azacytidine (AZA) or decitabine (DAC) have efficacy in sporadic TNBCs in vitro and in vivo. PARPi act not only to catalytically inhibit PARP but also to trap PARP at DNA single strand breaks (SSB), leading to suicidal DNA-protein crosslinks (DPC). We also showed that Talazoparib in combination with AZA/DAC increases PARP trapping in DNA, leading to increased and persistent levels of lethal DSBs, suggesting that DSBR may also be impaired with this treatment. HR plays an important role in resolving DPC in mammalian cells and Fanconi anemia (FA)-dependent HR has been previously shown to resolve AZA-induced replication lesions. Herein, we determined whether HR is impaired with combination drug treatment using a chromosomally integrated GFP-based reporter in the TNBC cell line, MDA-MB-231. HR activity was significantly (p<0.05) decreased with AZA or AZA/Talazoparib combination treatment, suggesting that AZA mediates decreased HR activity. We next questioned whether subsets of HR genes are indirectly down-regulated by the epigenetic reprogramming effects of DNMTis, thereby contributing to the efficacy of DNMTi and PARPi combination therapy. Microarray analysis was performed in multiple TNBC cell lines (including MDA-MB-231, MDA-MB-468, and SUM159PT) post DNMTi treatment and showed significant (p<0.05) decreases in expression of FANCD2, FANCC, and FANCE that could potentially generate a synthetic lethality when combined with PARPi, as has recently been reported. Decreased FA gene expression was validated by qPCR of mRNA and western analysis of proteins. Moreover, treatment of TNBC with DNA crosslinking agents, which require FA-dependent repair, have increased sensitivity post DNMTi treatment. These data suggest that decreased FA gene expression contributes to the efficacy of PARPi/DNMTi treatment in TNBC. Work is now underway to determine whether depletion of FA proteins can increase PARP trapping in PARPis treated TNBCs, leading to increased levels of cytotoxic DSBs. Citation Format: Lena J. McLaughlin, Huili Li, Pratik Nagaria, Stephen B. Baylin, Cynthia A. Zahnow, Feyruz V. Rassool. Decreased Fanconi anemia gene expression contributes to efficacy of PARP and DNMT inhibitor combination therapy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4045. doi:10.1158/1538-7445.AM2017-4045

Abstract 1579: Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate

Abstract Hexavalent chromium [Cr(VI)]-containing compounds are human carcinogens. They cause cancers in the respiratory system when inhaled, and stomach/other internal cancers when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induced base substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA-DNA cross links and DNA-protein cross-links in mammalian cells. We examined the ability of the soluble chromium compounds, sodium chromate (Na2CrO4), calcium chromate (CaCrO4) and potassium chromate (K2CrO4) to induce cytotoxicity and morphological transformation in C3H/10T½ Cl 8 (10T1/2) mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and dehydroascorbate, can reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III) intracellularly, making it a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in the mM range under physiological conditions in humans, but is only present in the μM range in mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum. We hypothesized the relatively weak responses for induction of morphological transformation of mammalian cells by Cr(VI) compounds in culture (dose-dependent but weak induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium chromate, and sodium chromate) is due to the small amounts of ascorbate in cultures of mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore, we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects of the highest non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T½ cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed that ascorbate itself exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and ascorbate, ascorbate played dual roles. It served as a pro-oxidant (enhancer of the cytotoxicity of chromate) at concentrations up to 0.1 mM, and as an anti-oxidant (reducer of the cytotoxicity of chromate) at concentrations of 0.25 mM and higher. This information is important for our ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. We project that at concentrations of 0.1 mM and lower, ascorbate will enhance the cell transforming activity of Cr(VI) compounds. Supported by undergraduate fellowships from the Provost's Office at the University of Southern California (USC) (P. I., JRL), by funding from the M. S. Program in Molecular Microbiology and Immunology to JRL, by discretionary funding to JRL, and by prior grant ES03341 from NIEHS/NIH (P. I., JRL). Citation Format: Sophia A. Shahin, William Liao, Laureen Tran, Qasim A. Akinwumi, Farn-shuan Tseng, Alyssa Mathew-Joseph, Joseph R. Landolph. Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2014-1579

Homologous Recombination but Not Nucleotide Excision Repair Plays a Pivotal Role in Tolerance of DNA-Protein Cross-links in Mammalian Cells

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells

Arsenic is generally recognized as a nonmutagenic carcinogen because sodium arsenite induces DNA damage only at very high concentrations. In this study we demonstrate that arsenite concentrations above 0.25 μM induce DNA strand breaks in both human leukemia cells and Chinese hamster ovary cells. Therefore, DNA damage may be involved in arsenic-induced carcinogenesis. Formamidopyrimidine-DNA glycosylase and proteinase K greatly increased DNA strand breaks in arsenite-treated cells, providing evidence that a large portion of arsenite-induced DNA strand breaks come from excision of oxidative DNA adducts and DNA-protein cross-links. Because DNA strand breaks appear only temporarily during excision repair, the level of detectable DNA strand breaks will be low at any given time point. For this reason many previous studies have only detected low levels of DNA strand breaks. We also show that catalase, and inhibitors of calcium, nitric oxide synthase, superoxide dismutase, and myeloperoxidase, could modulate arsenite-induced DNA damage. We conclude that arsenite induces DNA adducts through calcium-mediated production of peroxynitrite, hypochlorous acid, and hydroxyl radicals.

Formation of DNA-protein cross-links in mammalian cells by levuglandin E2.

Levuglandin E2 (LGE2), a rearrangement product derived from the prostaglandin endoperoxide, PGH2, causes repair-resistant DNA-protein cross-links and cell death (LD50 = 230 nM) in V79 Chinese hamster lung fibroblasts. The half-life for sequestration of LGE2 by covalent binding to cellular nucleophiles is at least an hour for 10 microM LG. This suggests that the in vivo production and distribution of free LGs should be measurable on this time scale. Following removal of the LGE2 and the return of the cultures to normal growth medium, additional DNA-protein cross-links continued to form over the ensuing 6-24 h. The results suggest that LG adducts to DNA or protein are not repaired, but react further at sites on protein or DNA in close proximity to the initial adducts, forming cross-links in a slow phase of the process.

A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells

Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diamminedichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 μM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: (A) Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4–6 days, the approximate doubling time of the cell population. (B) Either “used” culture medium or cycloheximide (1 μg/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01–5 μg/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. (C) The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. (D) In the presence of aphidicolin (10 μg/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. (E) Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled DNA indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of DNA replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is linked with cell cycling.

Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation

To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 m m sodium hydroxide (0.33 ml/min) instead of the standard 20 m m EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 μ m, trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.

Repair of deoxyribonucleic acid lesions caused by adriamycin and ellipticine

The intercalating agents adriamycin and ellipticine caused DNA single and double strand breaks and DNA-protein crosslinks in mammalian cells. Ellipticine caused a much higher frequency of each of these lesions than adriamycin, at equitoxic doses. However, adriamycin-induced DNA effects were slowly and incompletely repaired over 24 hr after drug removal while the effects of ellipticine were virtually completely repaired within 30 min. Repair of ellipticine-induced lesions was observed only if cells were diluted (~100-fold) after drug treatment, suggesting that significant amounts of drug may have been sequestered in the cellular membranes. Cell dilution did not alter the effects of adriamycin. Removal of the DNA lesions was temperature dependent. Finally, the similarity in the rates of repair for DNA single and double strand breaks and DNA-protein crosslinks supports the hypothesis that they are functionally related.

DNA-protein cross-linking by the mycotoxin, botryodiplodin, in mammalian cells.

Botryodiplodin, a mycotoxin isolated from a strain of Penicillium roqueforti, induces DNA-protein cross-links in mammalian cells. This action depends upon the presence of the hydroxyl group at the position 2 of the molecule. The mechanism involved in the formation of cross-linkings is discussed.