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

Abstract

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.Significance StatementCtIP helps to repair broken chromosomes through its ability to bind and bridge DNA molecules. We studied the structural and biochemical basis for these activities and how they are affected by hereditary CtIP mutations associated with developmental disorders. We discovered a minimal domain in the C-terminal region of CtIP which supports DNA binding as a monomer. DNA binding is non-specific and facilitates 1D diffusion, but binding alone is insufficient for intermolecular tethering of DNA molecules which requires tetramerization of CtIP via N-terminal coiled-coil domains. All disease variants tested displayed impaired DNA bridging activity. These results have important implications for understanding the role of CtIP as a hub protein for DNA break repair and its dysfunction in human disease.