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J Biol Chem. 2019 Mar 1;294(9):3312-3320. doi: 10.1074/jbc.RA118.006759. Epub 2019 Jan 9.

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

Author information

1
From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709.
2
the Department of Physics and Astronomy, University of North Carolina, Chapel Hill, North Carolina 27695, and.
3
the Department of Chemistry, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 derie@unc.edu.
4
From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, williamsrs@niehs.nih.gov.

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.

KEYWORDS:

CtIP; Ctp1; DNA bridging; DNA repair; DNA-binding protein; Sae2; Schizosaccharomyces pombe; atomic force microscopy (AFM); chromosome damage; homologous recombination; structural biology

PMID:
30626735
PMCID:
PMC6398140
DOI:
10.1074/jbc.RA118.006759
[Indexed for MEDLINE]
Free PMC Article

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