One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 59-39 exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replicationassociated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A-and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.
Background: The nucleases hSNM1A and hSNM1B are implicated in DNA interstrand cross-link repair.Results: hSNM1A and hSNM1B were biochemically characterized using undamaged and cross-linked DNA. A real-time assay for the nucleases suitable for inhibitor identification was developed.Conclusion: Preferential hSNM1A activation by high molecular weight and cross-linked DNA was observed.Significance: This work provides a basis for hSNM1A inhibitor development for improved cancer therapy.
DNA damaging agents have been widely used in cancer chemotherapy for many years and have proved successful in the treatment of both solid tissue and haematological malignancies. Many commonly used clinical agents, such as members of the nitrogen mustard, chloroethylnitrosourea, dimethane-sulphonate and platinum classes, are bifunctional. DNA interstrand crosslinks (ISC) formed in cells are clearly critical cytotoxic lesions and the formation of DNA ISC has been shown to correlate with cytotoxicity in vitro (1-5). Acquired resistance in vitro to such agents can occur by a number of mechanisms, for example altered drug transport (6), intracellular detoxification via enhanced glutathione and glutathione-S-transferase activity (7), but enhanced DNA repair capacity can also play an important role (3). Clinically the mechanisms of acquired resistance to DNA damaging agents are less clear but enhanced repair of ISC has been suggested to play a role in the acquired resistance of some cancers, e.g., chronic lymphocytic leukaemia to nitrogen mustards (8). In addition, the inherent sensitivity (and curability) of some tumors, e.g., testicular cancer, to DNA damaging agents may result in part from their inability to repair critical DNA lesions (9).
The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.
The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials.
A number of new ionic titanocene compounds have been isolated and characterised, which exhibit excellent cytotoxicity against different human tumour cell lines including a defined cisplatin resistant cell line. A range of biological assays have been carried out to determine levels of cytotoxicity and levels of DNA interstrand crosslinking.
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