Since the initial identification of DNA ligase inhibitors by

Since the initial identification of DNA ligase inhibitors by a structure-based approach [[18], [26]], there have been several reports describing LigI inhibitors using computer modelling and derivatives of the original DNA ligase inhibitors [[49], [50], [51]]. While these studies have shown that the inhibitors have activity against LigI in vitro, their affinity and selectivity appears to be less than that of L82-G17. Furthermore, there is no definitive evidence that these inhibitors target LigI in cells. Here we have shown that LIG1 null STF-62247 receptor are more resistant to L82-G17, presumably because there is no formation of trapped LigI-DNA complexes. Furthermore, cells that lack nuclear LigIIIα are more sensitive to L82-G17 as they lack a back-up activity for DNA replication. The elevated levels of LigI in cancer cells [[17], [18]] and the apparent viability of mammalian cells that lack LigI [[2], [33], [34]] suggest that LigI-selective inhibitors may preferentially target cancer cells because of their high proliferation rate. Toxicity in normal cells is likely be limited because of the ability of LigIIIα to substitute for LigI in DNA replication and repair [[6], [11]]. In the structure-activity studies described here, we have identified and characterized a novel uncompetitive inhibitor of LigI that selectively targets LigI both in in vitro and in cells. Further work to develop improved uncompetitive inhibitors would be facilitated by the determination of atomic resolution structures of inhibitor-bound LigI-DNA complexes [46]. Alternatively, the predicted binding site for the LigI inhibitors could be validated using site-directed mutagenesis to generate versions of LigI that retain wild type catalytic activity but are resistant to the inhibitors.
Materials & methods
Conflict of interest
Acknowledgements We thank Dr. Jennifer Gillette for the use of her plate reader and Genevieve Phillips at the UNM Fluorescence Microscopy Shared Resource for her invaluable knowledge and assistance. This work was supported by the University of New Mexico Comprehensive Cancer Center (P30 CA118100) and National Institute of Health Grants R01 GM57479 (to A.E.T.) and P01 CA92584.
Introduction DNA double-strand breaks (DSBs) are genomic lesions that play an important role in human health and disease. They are frequently generated by exogenous damaging agents (e.g., ionizing radiation) or as programmed intermediates in meiosis and V(D)J recombination (Mehta and Haber, 2014). The ends generated by these biological sources of chromosome breaks are often “complex,” with DNA helix-distorting nucleotide damage, mismatches, or chemical adducts that pose challenges to the ligases and polymerases needed for DSB repair (Breen and Murphy, 1995, Nitiss, 2009, Roth et al., 1992). This problem is especially relevant to the nonhomologous end joining (NHEJ) pathway, since, unlike other DSB repair pathways, these complex ends are not extensively resected prior to synthetic steps (polymerase and ligase activity; Waters et al., 2014a). Ligation is the only essential step in NHEJ and is performed by one of the three mammalian ligases, DNA ligase IV (LIG4; Wilson et al., 1997). LIG4 is recruited to broken ends through participation in a complex of core NHEJ factors, including XRCC4, the Ku 70/80 heterodimer (Ku; Nick McElhinny et al., 2000), and XLF. This NHEJ core complex is sufficient to physically link a pair of broken ends together and can thus be termed the paired-end complex (PEC). The PEC is essential for repair of diverse end structures; for example, XLF is required both for stable PEC formation (Reid et al., 2015) and ligation of complex ends, but it only modestly affects ligation of ends with complementary termini (Andres et al., 2007, Gu et al., 2007, Tsai et al., 2007). Recent physical analyses of PECs indicate that they are highly dynamic (Reid et al., 2015) and that both the flexibility and stability of PECs can be modulated by ligation-compatible DNA end chemistry (Reid et al., 2017). However, it is unclear how differences in end structure trigger these changes in dynamics and whether these changes in dynamics impact cellular repair.