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  • Two parameters were extracted from these

    2020-03-13

    Two parameters were extracted from these experiments to compare the interactions of the three compounds with ct-DNA. The association constant K for the binding of the naphthalimide derivatives to ct-DNA was calculated by fitting the evolution of the fluorescence spectra with a 1:1 binding model using the program Specfit (B). The corresponding K values are reported in and exemplifies differences in the binding of the three compounds with a significantly larger affinity for the non-substituted thiazonaphthalimide (K = 10 M). The substitution of the thiazole ring by the mesomeric electron-donating amino group decreases the affinity for the double helix by a factor of 2–3.5. Moreover, compound also displays the largest fluorescence quenching upon ct-DNA binding. The melting temperature of double-stranded DNA can be used to partially characterize the mode of interaction of aromatic compounds with DNA. No significant alteration of the melting temperature was observed in the presence of thiazonaphthalimide compounds , and , presumably indicating that none of the three aromatic compounds interacts with DNA an intercalative process between the A 77636 hydrochloride pairs of DNA (see ). Indeed, although an intercalative process is mentioned in the literature for the interaction between thiazonaphthalimide derivatives and ct-DNA, little evidence has been provided. In summary, we have reported the synthesis and unambiguous characterization of angular thiazonaphthalimide derivatives. The formation of linear regioisomers was revealed to not be possible, which is in accordance with the poor reactivity of position 2 in the ring. Compounds , and strongly bind to ct-DNA, with no significant evolution of the ct-DNA melting temperature upon binding, which suggests a non-intercalative process. The largest affinity and fluorescence quenching is observed for the non-substituted ring. The next step will be to graft compound to amino groups found in peptides or proteins to obtain fluorescent probes sensitive to DNA. Acknowledgments The authors thank Pierre-Alain Bayle for his contribution in the recording of NMR data and Colette Lebrun for ESI-MS spectrum. This research was supported by the Labex ARCANE (Grant ANR-11-LABX-0003-01).
    Introduction The activation of oncogenes during tumorigenesis generates DNA replication stress (RS), which is a known driver of chromosomal instability (CIN) (Burrell et al., 2013, Macheret and Halazonetis, 2015). Certain regions of the human genome, such as common fragile sites (CFSs), are particularly sensitive to RS. These loci are difficult to replicate and are prone to generate copy number variations (CNVs) and chromosomal rearrangements in human cancers (Arlt et al., 2006, Burrow et al., 2009, Wilson et al., 2015). There are several features that the different CFS loci often have in common, including a propensity to form DNA secondary structures within AT-rich sequences, the presence of a long primary transcript that can potentially impede replication fork progression, and increased levels of R-loops (Helmrich et al., 2011, Le Tallec et al., 2013, Santos-Pereira and Aguilera, 2015, Sarni and Kerem, 2016, Sollier and Cimprich, 2015, Zhang and Freudenreich, 2007). CFSs manifest as DAPI-negative gaps on metaphase chromosomes (CFS expression), a phenomenon that is exacerbated by exposing cells in S-phase to a mild dose of aphidicolin (APH) (Glover et al., 1984). CFS expression is an active process that requires the MUS81-EME1 DNA structure-selective endonuclease and its associated scaffold protein, SLX4 (Minocherhomji and Hickson, 2014, Minocherhomji et al., 2015, Naim et al., 2013, Ying et al., 2013). In response to RS, the FANCD2/FANCI protein complex forms “twin foci” (one on each sister chromatid) at CFS loci that can persist into mitosis (Chan et al., 2009, Howlett et al., 2005, Minocherhomji et al., 2015). These foci co-localize with SLX4 and serve as a useful surrogate marker of the location of CFSs in G2/M phase cells. Upon mitotic entry, CDK1-dependent phosphorylation of EME1 promotes the association of MUS81-EME1 with SLX4 at under-replicated CFS loci (Minocherhomji et al., 2015, Wyatt et al., 2013, Ying et al., 2013). An important function of MUS81-EME1 at CFSs is to promote DNA repair synthesis that occurs in the prophase of mitosis (Minocherhomji et al., 2015). This mitotic DNA synthesis (henceforth termed “MiDAS”) requires MUS81-EME1, SLX4, and a non-catalytic subunit of DNA polymerase δ, POLD3 (Pol32 in yeast) (Bursomanno et al., 2015, Minocherhomji et al., 2015). Figure 1A summarizes the previously proposed roles of these factors. DNA synthesized via the MiDAS pathway is associated with the appearance of cytogenetically defined DAP1-negative gaps on metaphase chromosomes at CFS loci (Minocherhomji et al., 2015). In the absence of MiDAS, potentially lethal chromosome missegregation occurs, and daughter cells exhibit an increased frequency of prominent structures termed 53BP1 nuclear bodies (comprising OPT domains), which are known to associate with missegregated CFSs (Harrigan et al., 2011, Lukas et al., 2011, Minocherhomji et al., 2015, Naim et al., 2013, Ying et al., 2013).