CtIP/Ctp1/Sae2, molecular form fit for function

Abstract

Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By modulating the nucleolytic 5'-3' resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein-protein interactions and DNA binding.

Keywords: CtIP/Ctp1/Sae2; DNA bridging; Homologous recombination; Intrinsically disordered proteins; Resection.

Figure 1

Initiation of homologous recombination. DNA double-strand breaks (DSBs) often contain “dirty” ends, with secondary DNA structure, protein and chemical adducts. Mre11-Rad50-Nbs1 (MRN) recognizes DNA breaks, bridging both across the DSB and to the sister chromatid. Mre11 is stimulated by Ctp1 and carries out a two-step resection, utilizing first endonuclease activity, then 3′-5′ exonuclease activity, generating single-stranded 3′-overhangs. These ssDNA overhangs are further resected and then bound by Rad51, forming a nucleoprotein filament for invasion of the sister chromatid, initiating homologous recombination repair.

Figure 2

Alignment of predicted structural and conserved domains of Ctp1 (S. pombe), CtIP (H. sapiens) and Sae2 (S. cerevisiae). Predictions of protein structure (white) and disorder (grey) were generated by D²P² [40], with regions corresponding to the THDD and SAE2-like domain noted above. Corresponding regions of conserved function and structure (colored boxes) are aligned below the structural prediction. Key phosphorylation sites of Ctp1 and its homologs are denoted as yellow circles.

Figure 3

Structural features of Ctp1 and CtIP. (A) Domain organization of Ctp1 (S. pombe) and CtIP (H.sapiens). The domains of Ctp1 (orange schematic) highlight the core N-terminal tetrameric helical dimer of dimers (THDD), an intrinsically disordered region that contains both the phosphorylated pS-x-pT motif for Nbs1-binding (green dotted box) and the C-terminal SAE2-like domain. Crystal structures of Ctp1 (orange; PDB 4X01) and CtIP (blue; PDB 4D2H) show the conserved tetramerization core formed by interlocking alpha-helices. (B) Comparison of Ctp1 and CtIP tetramerization. Overlay of Ctp1 (orange/yellow) and CtIP (blue/gray) tetramerization interface, mediated by leucine and aromatic amino acids. (C) DNA binding surfaces. Ctp1-DNA interactions map to the exposed surface of Ctp1 dimers (orange). A similar basic surface in CtIP also exists. (D) Ctp1-Nbs1 complex. The phosphorylated Ctp1 (orange) binds a positively charged, surface-exposed phosphoprotein recognition pocket of the Nbs1 FHA domain (green) (PDB 3HUF).

Figure 4

Functional roles for Ctp1 in homologous recombination repair. (A) Biochemical activities of Ctp1 and Ctp1 homologs. Ctp1 and Sae2 act as bridging factors between two DNA molecules, while Sae2 and CtIP are co-factors of MRN, which stimulate Mre11 endonuclease activity at protein blocked DNA ends. Ctp1 lacks nuclease activity [5], and reports of endonuclease activity on forked and hairpin DNA structures by Sae2 and CtIP are inconsistent [17,18,74,76,89,91]. Arrows mark reported endonuclease cut sites. (B) Model of Ctp1 bridging DNA. Ctp1 contains multiple DNA binding sites and contains inherent flexibility in its intrinsically disordered region. Different bridging architectures are possible, with one potential bridging mode depicted here. (C) Model of MRN and Ctp1 at a protein-blocked DNA DSB. Mre11 harbors endonuclease activity, while Nbs1 links Ctp1 to the MRN complex. Ctp1 bridges across the DNA double-strand break, facilitating coordinated resection.

Figure 5

Regulation of Ctp1/CtIP/Sae2. (A) Protein expression levels of Ctp1 and its homologs are upregulated during S and G2 phase of the cell cycle, coincident with active homologous recombination repair. Regulation of Ctp1/CtIP/Sae2 activity during repair also relies on post-translational modifications. Sae2 and CtIP phosphorylation is required for HR, while acetylation targets CtIP for degradation. (B) Alignment of conserved C-terminal SAE2-like domain. Ser267 in S. cerevisiae is critical for cell-cycle regulation of DSB repair, yet is not conserved in S. pombe. Conserved residues are highlighted grey.