Ctp1 protein-DNA filaments promote DNA Bridging and DNA double-strand break repair: 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 Mre11Rad50Nbs1 nuclease complex activity, and harbor DNA-binding and -bridging activities. However, the molecular basis for Ctp1DNA transactions remain undefined. Here, we report atomic force microscopy (AFM) imaging of S. pombe Ctp1DNA 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 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. Two-Tiered Enforcement of High-Fidelity DNA Ligation: DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3'-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg2+-reinforced DNA binding to validate DNA base pairing during the adenylyl-transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg2+, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage. Ubiquitin Stimulated Reversal of Topoisomerase 2 DNAProtein Crosslinks by TDP2: TyrosylDNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds Ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 polyUbiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-Ubiquitin chains of greater than Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle Xray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves UBA-regulated autoinhibitory states of TDP2. Our data indicates that that polyUb regulates TDP2catalyzed TOP2DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.
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| Tumbale, Percy; Schellenberg, Matthew J; Mueller, Geoffrey A et al. (2018) Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease. EMBO J 37: |
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| Andres, Sara N; Williams, R Scott (2017) CtIP/Ctp1/Sae2, molecular form fit for function. DNA Repair (Amst) 56:109-117 |
| Wallace, Bret D; Berman, Zachary; Mueller, Geoffrey A et al. (2017) APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress. Proc Natl Acad Sci U S A 114:304-309 |
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| Appel, C Denise; Feld, Geoffrey K; Wallace, Bret D et al. (2016) Structure of the sirtuin-linked macrodomain SAV0325 from Staphylococcus aureus. Protein Sci 25:1682-91 |
| Andres, Sara N; Appel, C Denise; Westmoreland, James W et al. (2015) Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair. Nat Struct Mol Biol 22:158-66 |
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