It is currently controversial as to whether purified CtIP and its orthologues possess intrinsic nuclease activity (Andres and Williams, 2017). Therefore, to test our CtIP preparations for nuclease activity we incubated CtIP at high concentrations with a forked DNA substrate in the presence of various divalent cations and for extended times. (A) Representative nuclease assay for a wild type CtIP preparation (concentrations as indicated in (B)) incubated with 5 nM 5′-Cy5-labelled forked DNA for two hours. (B) Quantified nuclease assays using several different wild type CtIP preparations (labelled A-C) and different CtIP mutants (as indicated) titrated against 5 nM forked DNA. (C) Representative nuclease assay using 250 nM wild type CtIP against 5 nM forked DNA in the presence of different divalent cations. (D) Quantified nuclease assay using 250 nM wild type CtIP against 5 nM forked DNA in the presence of different divalent cations. Nuclease activity was observed which was consistent with cleavage of the 5′-terminated ssDNA flap as reported previously. This nuclease activity may explain the small proportion of less than single contour length substrates observed in our AFM bridging experiments. However, at the highest concentrations tested (which are >> Kd for the binding interaction with the DNA) the half-life of the DNA fork substrate was on the order of several hours. The specific nuclease activity varied between wild type preparations and did not correlate with the ability of CtIP to bind DNA. For example, the C-terminal deletion mutant CtIP1-782 does not bind DNA (data not shown) while dephosphorylated CtIP has ~ 10 fold greater affinity for DNA than wild type (Figure 4, main text), but both preparations retain apparent nuclease activity comparable to wild type. Mutations designed to inactivate a putative nuclease active site (N289A and H290A) did reduce the observed nuclease activity in the preparation as reported previously, but a mutation thought to result in hyperactivated nuclease activity (S347D) was less active than any of our wild type preparations (Makharashvili et al., 2014). The dependence of the observed activity on divalent cation identity was qualitatively different for our preparation compared to that reported previously, with Mg2+ being the marginally favoured co-factor as opposed to a substantial preference for Mn2+ (Makharashvili et al., 2014). Taken together, these observations suggest to us that the nuclease activity in the preparation most likely belongs to a contaminant or co-purified protein rather than to the CtIP polypeptide. However, we are unable to exclude the possibility that CtIP (either as prepared or dephosphorylated) possesses a very weak and/or tightly-regulated nuclease activity.