Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair

Abstract

Ctp1 (aka CtIP or Sae2) collaborates with Mre11–Rad50–Nbs1 to initiate repair of DNA double strand breaks (DSBs), but its function(s) remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 harbors multivalent DNA-binding and bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal “RHR” DNA interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA bridging activity in vitro and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles for Ctp1 in binding and coordination of DSB repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CTIP-linked Seckel and Jawad syndromes.

DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination

“…Sae2 itself was also shown to possess a nuclease activity specific to secondary structures in DNA (60), although an enzymatic activity was not detected by other laboratories (27,50). Human and Schizosaccharomyces pombe Sae2 homologues (CtIP and Ctp1, respectively) were found to tetramerize, which was shown to be important for their function in vivo (61,62). Similarly, mutations that prevent oligomerization of Sae2 in vivo resulted in null phenotypes in several genetic assays (53).…”

When two is not enough: a CtIP tetramer is required for DNA repair by Homologous Recombination

“…Our own new data, 19 together with the work by Andres and colleagues, 20 has now solved this issue for human CtIP and fission yeast Ctp1, respectively. Both reports provide biophysical and structural evidence that CtIP and Ctp1 exist as constitutive tetramers and share a mode of self-association that has remained remarkably conserved over a billion years of evolutionary history.…”

“…33,34 However, L27E CtIP shows no defect in its interaction with either NBS1 19 or FANCD2 (our unpublished data). We note that the conserved C-terminal domain (CTD) of CtIP and Ctp1 can bind DNA 19,20 and that mutation of Ctp1 residues critical for DNA binding causes hypersensitivity to DNA-damaging agents. 20 The tetrameric architecture adopted by CtIP/Ctp1 thus appears well suited to simultaneously position multiple CTDs on distinct DNA molecules.…”

“…We note that the conserved C-terminal domain (CTD) of CtIP and Ctp1 can bind DNA 19,20 and that mutation of Ctp1 residues critical for DNA binding causes hypersensitivity to DNA-damaging agents. 20 The tetrameric architecture adopted by CtIP/Ctp1 thus appears well suited to simultaneously position multiple CTDs on distinct DNA molecules. Although a CtIP tetramer could in principle locate its CTDs on 4 different DNA molecules, we favor the possibility that a 'dimer of dimers' CtIP could locate pairs of CTDs at the 2 ends of a DNA DSB, 20 in preparation for simultaneous processing of DNA ends (Fig.…”

“…19,20 The new evidence uncovers a remarkable oligomerisation mechanism that is shared by human CtIP and its fission-yeast ortholog Ctp1: juxtaposition of their conserved N-terminal regions results in a specific tetrameric structure that is necessary for effective DSB repair by HR.…”