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  • Although high concentrations of SCR G and SCR R inhibited


    Although high concentrations of SCR7-G and SCR7-R inhibited LigIV as reported [11], the SCR7 derivatives were more effective inhibitors of LigIIIα and, in particular, LigI. Although it is possible that differences in the DNA substrate underlie this discrepancy, it appears more likely that this reflects actual differences in how the compounds affect the different ligases. In the published study [11], multiple other bands were present in the DNA ligase preparations purified from Escherichia coli in addition to the DNA ligase polypeptides and protein partners identified by immunoblotting [11]. In contrast, the fractions of DNA LigIIIα/XRCC1 and LigIV/XRCC4 used in our study were purified after expression in insect cells, which permits higher and more reliable enzyme activity. While it was reported that SCR7-R inhibited the joining of linear DNA molecules by extracts from rat testes [11], it has been established that extracts from mammalian cell lines have robust end joining activity that, in most cases, is not dependent on the core factors involved in the major NHEJ pathway [25], [26], [27], [28]. Unlike the extract assay developed by Baumann and West [29], there is no evidence that the end joining carried out by the rat testes extract is dependent upon LigIV and the other core NHEJ factors. It was also reported that SCR7 reduced the frequency of NHEJ in cell-based assays with plasmid substrates. Notably, these assays used concentration of SCR7-R that were 10-fold lower than the concentration of SCR7-R used to inhibit purified DNA ligase IV by 50% in vitro. In our study, we have used a cellular plasmid-based quantitative assay for V(D)J recombination that has been shown by many laboratories to be dependent upon LigIV [23], [24]. Concentrations of up to 200μM SCR7-R failed to reduce LigIV-dependent V(D)J recombination. This is in contrast to the less direct assay for V(D)J recombination in lymphoid cellsused by Raghavan et al., which also reduced the numbers of B and T arn 509 in mice treated with SCR7-R, suggesting toxicity [11]. It should be noted that there are many possible steps at which a compound might affect a PCR readout for V(D)J recombination in an animal model. In contrast, the cellular plasmid-based assay provides a direct and quantitative measure of LigIV-dependent V(D)J recombination. In summary, both SCR7-R and its closely related derivative SCR7-G are weak inhibitors of LigIV and exhibit higher activity against LigI and LigIIIα/XRCC1. Furthermore, SCR7-R did not inhibit a V(D)J recombination assay that is dependent upon LigIV. Recent studies have shown that either knockdown of a core NHEJ factor [30] or incubation with SCR7-X [30], [31] increases the efficiency of CRISPR-Cas9-induced precise genome editing. Since SCR7-X/SCR7-G enhanced CRISPR-CAS9 [30], [31] at a concentration of SCR7-X/SCR7-G (1μM) that was significantly lower than the concentration required to inhibit LigIV in vitro (>200μM), it appears likely that SCR7-X/SCR7-G is causing this effect by mechanisms other than inhibition of LigIV.
    Acknowledgements We thank Dr. Sathees Raghavan for the sample of SCR7-R. This work was supported by US National Institute of Health Grants, R01 GM47251, GM57479, ES01252 and P01CA92584, to AET) and by US National Institute of Health funding to MRL. Flow cytometry and microscopy were carried out in University of New Mexico Cancer Center Shared Resources supported by NCI Cancer Center Support Grant P30CA11800.
    Introduction DNA ligases are enzymes present in almost all forms of life. Besides bacteria, eukaryotes and archaea, a number of viruses carry genes encoding for their functional ligases [1], [2], [3]. To date, various DNA ligases have been discovered and characterized in respect to their molecular size and type of required cofactors. Based on cofactor preferences, the ligase family is divided into ATP- and NAD+-dependent enzymes. The bacteriophage, archaea and most of the eukaryotic DNA ligases belong to the first group of enzymes, whereas the NAD+-dependent group includes mainly bacterial enzymes [2].