br Materials and methods br Results br Discussion Raghavan a

Materials and methods
Results
Discussion Raghavan and colleagues described the synthesis and characterization of a compound, SCR7, that inhibited DNA ligase IV and blocked DNA ligase IV-dependent NHEJ in extracts and cell-based assays [11]. Furthermore, they reported that this GW4064 receptor reduced the growth of tumor xenografts, both in combination with genotoxic agents used clinically and as a single agent [11]. In this study, we have found that the published synthesis protocol does not generate the compound with the structure described by Raghavan and colleagues and that SCR7 provided by Dr. Raghavan (SCR7-R) also does not have the structure described [11]. We have determined the structure of SCR7-R and SCR7-G, the compounds generated by the synthesis protocol described by Raghavan and colleagues [11]. Furthermore, our analysis of commercially available SCR7 (from XcessBio and designated SCR7-X here) revealed that it has the same structure as SCR7-G rather than the structure listed by XcessBio. 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 cells 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.