Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair.

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis.

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation 'switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation.

Activation of the γ-tubulin complex by the Mto1/2 complex.

The multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells, but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a "minimal" version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes.

The RecQ4 orthologue Hrq1 is critical for DNA interstrand cross-link repair and genome stability in fission yeast.

Of the five human RecQ family helicases, RecQ4, BLM, and WRN suppress distinct genome instability-linked diseases with severe phenotypes, often with indeterminate etiologies. Here, we functionally define Hrq1, a novel orthologue of RecQ4 from fission yeast. Biochemical analysis of Hrq1 reveals a DEAH box- and ATP-dependent 3'-5' helicase activity on various DNA substrates, including bubbles but not blunt duplexes, characteristic of the RecQ family. Cells lacking Hrq1 suffer spontaneous genomic instability and, consequently, require homologous recombination repair and the DNA damage checkpoint for viability. Hrq1 supports the nucleotide excision repair of DNA damage caused by the chemotherapeutic agent cisplatin and, in certain genetic contexts, UV light. Genetic epistasis analyses reveal that Hrq1 acts parallel to the PCNA/Ubc13/Mms2-dependent postreplication repair (PRR) pathway. Thus, in hrq1Δ cells, lesions are channeled through the PRR pathway, yielding hyper-recombinant and mutator phenotypes; analogous defects may underlie the genetic instability and diseases associated with RecQ4 dysfunction.

Two distinct regions of Mto1 are required for normal microtubule nucleation and efficient association with the gamma-tubulin complex in vivo.

Cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe involves the interacting proteins Mto1 and Mto2, which are thought to recruit the gamma-tubulin complex (gamma-TuC) to prospective microtubule organizing centres. Mto1 contains a short amino-terminal region (CM1) that is conserved in higher eukaryotic proteins implicated in microtubule organization, centrosome function and/or brain development. Here we show that mutations in the Mto1 CM1 region generate mutant proteins that are functionally null for cytoplasmic microtubule nucleation and interaction with the gamma-TuC (phenocopying mto1Delta), even though the Mto1-mutant proteins localize normally in cells and can bind Mto2. Interestingly, the CM1 region is not sufficient for efficient interaction with the gamma-TuC. Mutation within a different region of Mto1, outside CM1, abrogates Mto2 binding and also impairs cytoplasmic microtubule nucleation and Mto1 association with the gamma-TuC. However, this mutation allows limited microtubule nucleation in vivo, phenocopying mto2Delta rather than mto1Delta. Further experiments suggest that Mto1 and Mto2 form a complex (Mto1/2 complex) independent of the gamma-TuC and that Mto1 and Mto2 can each associate with the gamma-TuC in the absence of the other, albeit extremely weakly compared to when both Mto1 and Mto2 are present. We propose that Mto2 acts cooperatively with Mto1 to promote association of the Mto1/2 complex with the gamma-TuC.