DNA topoisomerases carry out topological transformations of DNA by introducing transient DNA breaks. The covalent intermediate of topoisomerase reactions include the topoisomerase protein covalently bound to DNA by a phosphotyrosine intermediate. Anti-cancer drugs that target topoisomerases typically trap the covalent intermediate, and generate cytotoxic enzyme dependent DNA damage. More recently, structural alterations in DNA such as DNA damage have also been shown to trap covalent intermediates of topoisomerase reactions. Understanding the action of drugs that target topoisomerases as well as determining the importance of trapped topoisomerases on genome stability requires assays that can accurately and sensitively measure levels of topoisomerase/DNA complexes. This chapter describes two approaches that have been developed to quantitate topoisomerase DNA complexes. These assays termed ICE (in vivo complex of enzymes) and RADAR (rapid approach to DNA adduct recovery) rely on isolation of genomic DNA under conditions that preserve proteins covalently bound to DNA. Covalently bound proteins are then quantitated using antibodies directed against specific topoisomerases. We describe assays in both mammalian cells and the yeast Saccharomyces cerevisiae that can measure topoisomerase/DNA covalent complexes, and give examples that can be used to enhance the quantitative reliability of these assays.

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DNA damage tolerance and mutagenesis are hallmarks and enabling characteristics of neoplastic cells that drive tumorigenesis and allow cancer cells to resist therapy. The ‘Y-family’ trans-lesion synthesis (TLS) DNA polymerases enable cells to replicate damaged genomes, thereby conferring DNA damage tolerance. Moreover, Y-family DNA polymerases are inherently error-prone and cause mutations. Therefore, TLS DNA polymerases are potential mediators of important tumorigenic phenotypes. The skin cancer-propensity syndrome xeroderma pigmentosum-variant (XPV) results from defects in the Y-family DNA Polymerase Pol eta (Polη) and compensatory deployment of alternative inappropriate DNA polymerases. However, the extent to which dysregulated TLS contributes to the underlying etiology of other human cancers is unclear. Here we consider the broad impact of TLS polymerases on tumorigenesis and cancer therapy. We survey the ways in which TLS DNA polymerases are pathologically altered in cancer. We summarize evidence that TLS polymerases shape cancer genomes, and review studies implicating dysregulated TLS as a driver of carcinogenesis. Because many cancer treatment regimens comprise DNA-damaging agents, pharmacological inhibition of TLS is an attractive strategy for sensitizing tumors to genotoxic therapies. Therefore, we discuss the pharmacological tractability of the TLS pathway and summarize recent progress on development of TLS inhibitors for therapeutic purposes.

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The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

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