Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair

Sara N Andres,Zimeng M Li,Dorothy A Erie,R Scott Williams

doi:10.1074/jbc.ra118.006759

Abstract

The Ctp1 protein in Schizosaccharomyces pombe is essential for DNA double-strand break (DSB) repair by homologous recombination. Fission yeast Ctp1 and its budding yeast (Sae2) and human (CtIP) homologs control Mre11-Rad50-Nbs1 nuclease complex activity and harbor DNA-binding and -bridging activities. However, the molecular basis for Ctp1-DNA transactions remains undefined. Here, we report atomic force microscopy (AFM) imaging of S. pombe Ctp1-DNA complexes revealing that Ctp1 polymerizes on dsDNA molecules and forms synaptic filaments that bridge two dsDNA strands. We observed that Ctp1 DNA filaments are typified by an average filament length of ∼180 bp of dsDNA and a Ctp1 tetramer footprint of ∼15 bp. Biochemical results characterizing Ctp1 variants with impaired DNA-binding or -bridging properties were consistent with Ctp1-mediated DNA bridging requiring the intact and correctly folded Ctp1 tetramer. Furthermore, mutations altering Ctp1 oligomerization and DNA bridging in vitro conferred cell sensitivity to DSB-producing agents. Together, these results support an important role for Ctp1-regulated DNA strand coordination required for DNA DSB repair in S. pombe.

Highlights

The Ctp1 protein in Schizosaccharomyces pombe is essential for DNA double-strand break (DSB) repair by homologous recombination
Based on a standard curve of atomic force microscopy (AFM) volume versus molecular weight, this volume corresponds to a molecular weight of 124 kDa (4 Ctp1 molecules) (28 –30), which is consistent with Ctp1 (Mr ϳ132 kDa) being predominantly tetrameric (Fig. S1C), consistent with multi-angle light scattering experiments [18]
Ctp1 is a critical component of the DNA DSB repair machinery, engaging with the Mre11–Rad50 – Nbs1 (MRN) complex to initiate resection of the DNA DSB (4 –6); the mechanism of Ctp1 action in this role is poorly defined

Summary

Introduction

The Ctp protein in Schizosaccharomyces pombe is essential for DNA double-strand break (DSB) repair by homologous recombination. We report atomic force microscopy (AFM) imaging of S. pombe Ctp1–DNA complexes revealing that Ctp polymerizes on dsDNA molecules and forms synaptic filaments that bridge two dsDNA strands. Mutations altering Ctp oligomerization and DNA bridging in vitro conferred cell sensitivity to DSB-producing agents. Together, these results support an important role for Ctp1-regulated DNA strand coordination required for DNA DSB repair in S. pombe. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Conclusion