Sarah C. Shuck

Eukaryotic nucleotide excision repair, from understanding mechanisms to influencing biology

Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER, while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

Targeted Inhibition of Replication Protein A Reveals Cytotoxic Activity, Synergy with Chemotherapeutic DNA-Damaging Agents, and Insight into Cellular Function

Targeting uncontrolled cell proliferation and resistance to DNA-damaging chemotherapeutics with a single agent has significant potential in cancer treatment. Replication protein A (RPA), the eukaryotic ssDNA-binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. We have identified a novel small molecule that inhibits the in vitro and cellular ssDNA-binding activity of RPA, prevents cell cycle progression, induces cytotoxicity, and increases the efficacy of chemotherapeutic DNA-damaging agents. These results provide new insight into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. This represents the first molecularly targeted eukaryotic DNA-binding inhibitor and reveals the utility of targeting a protein-DNA interaction as a therapeutic strategy for cancer treatment.

Identification of novel small molecule inhibitors of the XPA protein using in silico based screening

The nucleotide excision repair pathway catalyzes the removal of bulky adduct damage from DNA and requires the activity of more than 30 individual proteins and complexes. A diverse array of damage can be recognized and removed by the NER pathway including UV-induced adducts and intrastrand adducts induced by the chemotherapeutic compound cisplatin. The recognition of DNA damage is complex and involves a series of proteins including the xeroderma pigmentosum group A and C proteins and the UV-damage DNA binding protein. The xeroderma pigmentosum group A protein is unique in the sense that it is required for both transcription coupled and global genomic nucleotide excision repair. In addition, xeroderma pigmentosum group A protein is required for the removal of all types of DNA lesions repaired by nucleotide excision repair. Considering its importance in the damage recognition process, the minimal information available on the mechanism of DNA binding, and the potential that inhibition of xeroderma pigmentosum group A protein could enhance the therapeutic efficacy of platinum based anticancer drugs, we sought to identify and characterize small molecule inhibitors of the DNA binding activity of the xeroderma pigmentosum group A protein. In silico screening of a virtual small molecule library resulted in the identification of a class of molecules confirmed to inhibit the xeroderma pigmentosum group A protein-DNA interaction. Biochemical analysis of inhibition with varying DNA substrates revealed a common mechanism of xeroderma pigmentosum group A protein DNA binding to single-stranded DNA and cisplatin-damaged DNA.

Targeting the OB-Folds of Replication Protein A with Small Molecules

Replication protein A (RPA) is the main eukaryotic single-strand (ss) DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs) that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B) of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold) containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.

Selection of Monoclonal Antibodies Against 6-oxo-M1dG and Their Use in an LC-MS/MS Assay for the Presence of 6-oxo-M1dG in Vivo

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2′-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M1dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M1dG is oxidized to a primary metabolite, (3-(2-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M1dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M1dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M1dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([15N5]-6-oxo-M1dG) as an internal standard. Healthy male Sprague–Dawley rats excreted 6-oxo-M1dG at a rate of 350–1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M1dG in rodents without exogenous introduction of M1dG.

Targeting Nucleotide Excision Repair as a Mechanism to Increase Cisplatin Efficacy

Tumor resistance to chemotherapeutic DNA damaging agents, such as cisplatin, is an obstacle in the treatment of many cancers, including lung and ovarian. Resistance is influenced by nucleotide excision repair (NER) catalyzed removal of cisplatin-DNA lesions. NER is the primary pathway used by the cells in the repair of helix-distorting cisplatin lesions; therefore, inhibition of NER may increase the efficacy of cisplatin treatment. More specifically, the recognition and verification of DNA damage by NER is a critical step in the pathway, making it an ideal target for inhibition. Recognition of DNA damage occurs primarily through two proteins, Xeroderma Pigmentosum Group A (XPA) and replication protein A (RPA). XPA has been shown to have a role exclusively in NER, thus making it a highly specific target for inhibition that will lead to a decrease in NER and an increase in sensitivity to cisplatin treatment. RPA is a single-stranded DNA-binding protein that has roles in NER as well as in other metabolic pathways, including DNA replication and recombination. We have developed a high-throughput (HT) assay for XPA/RPA binding to DNA and screened libraries of small molecules to identify compounds capable of interrupting the protein/DNA interaction, an effort that has lead to the identification of small molecule inhibitors of both RPA and XPA. These inhibitors have been validated in secondary in vitro screens and structure—activity relationships were determined for one class of inhibitors. Further development of this class of compounds is anticipated to display cytostatic/cytotoxic activity and sensitize cells to cisplatin therapy.

Protein Modification by Adenine Propenal

Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine–lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA’s ability to bind to a DNA substrate.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N2-1-(carboxyethyl)-2′-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

Abstract A12: Alkylation of histones by 4-oxo-2-nonenal as a novel modification linked to oxidative stress

Sustained oxidative stress leads to the generation of toxic concentrations of the lipid aldehydes 4-hydroxy-2-nonenal (4-HNE) and 4-oxononenal (4-ONE), which are capable of covalently modifying the side-chains of Cys, His, and Lys residues. These protein modifications have been identified as a contributing factor in numerous disease states, including cancer, cardiovascular disease, neurodegeneration and diabetes. Here, we describe a novel class of histone modifications resulting from lipid electrophile adduction of His and Lys residues. Utilizing click chemistry with ω-alkynyl-4-ONE (a4-ONE), we have identified all-four core histones as targets for modification in RKO cells treated with physiologically relevant concentrations of electrophile. Isolation of chromatin from these cells reveals H2B as a highly susceptible target to modification by 4-ONE, while H2A, H3 and H4 show minimal reactivity. Interestingly, similar experiments with a4-HNE failed to identify any of the core histones as targets for modification. 4-HNE reacts heavily with Cys residues, which histones lack, whereas 4-ONE reacts primarily with the e-amine of Lys residues. A proteomic investigation of site-specific modifications in RKO cells treated with 4-ONE reveals H2B Lys117 as a major target for modification. The predominant stable species detected on H2BK117 results from a 1,2-addition and net acylation analogous to Lys acetylation. H2BK117 is a surface exposed Lys residing on the face of the nucleosome core particle. Treatment of RKO cells with physiologically relevant doses of 4-ONE results in the altered expression of 40 genes. Electrophile adduction of histones provides a previously unexplored link between oxidative stress and gene regulation. Citation Format: J J. Galligan, K L. Rose, W N. Beavers, C D. Aluise, S C. Shuck, L J. Marnett. Alkylation of histones by 4-oxo-2-nonenal as a novel modification linked to oxidative stress. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A12.