DNA binding and bridging by human CtIP in the healthy and diseased states.

The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed.

Modification of C-seco taxoids through ring tethering and substituent replacement leading to effective agents against tumor drug resistance mediated by ßIII-Tubulin and P-glycoprotein (P-gp) overexpressions.

In our efforts to improve the efficacy of taxane-based microtubule (MT) stabilizing agents against tumor drug resistance mediated by multiple mechanisms, two clinically relevant factors were focused: i.e., P-glycoprotein and ßIII-tubulin overexpression. Based on the structure of C-seco taxoid 1 m (IDN5390) which was believed to more selectively interact with ßIII-tubulin than paclitaxel, we prepared a series of C-seco taxoids bearing various 7,9-O-linkages and/or different substituents at C2 and C3' positions. Some of them exhibited much more potent binding affinity to MTs and cytotoxicity than their C-seco parent compounds in drug resistant cells with both mechanisms. SAR analysis indicated that C2 modifications significantly enhanced MT binding but brought ambiguous influence to cytotoxicity whereas 7,9-linkage and C3' modifications enhance cytotoxicity more efficiently than improve MT binding. These observations illustrate a better translation of molecular binding effect to cellular activity by C ring closure and C3' modification than C2 modification in C-seco taxoids.