The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

The Smc5/6 complex is involved in various DNA transactions and is best known for ensuring the fidelity of homologous recombination. We exploit single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association. We show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the two arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association. However, unlike a mutant attenuating chromatin association, a mutant that disrupts ssDNA binding results in highly elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.The structural maintenance of chromosomes (SMC) complexes cohesin, condensin and Smc5/6 are critical for the correct organisation of chromosome architecture 1 . Whereas the functions of cohesin and condensin are increasingly well understood, the Smc5/6 complex remains relatively ambiguous. Smc5/6 is conserved across all eukaryotes and is best known for its role in the cellular response to DNA damage by ensuring the fidelity of homologous recombination repair (HRR) 2,3 . Smc5/6 has been reported to promote replication fork stability 4 and facilitate DNA replication through natural pausing sites 5 . Biochemically, the complex can regulate prorecombinogenic helicases 6,7 . It has also been proposed to monitor DNA topology 8 and recently been shown to restrict viral transcription 9,10 . Complete inactivation of the Smc5/6 complex in a variety of organisms leads to cell death. However, hypomorphic mutants show significant defects in sister-chromatid HRR, display replication fork instability, are sensitive to a wide range of genotoxins and accumulate unresolved recombination intermediates 4,11,12 .Like all SMC complexes, the core of Smc5/6 is composed of two folded proteins, Smc5 and Smc6, which form a heterodimer ( Figure 1A). Each subunit comprises a long coiled-coil arm with a hinge region at one end and a globular ATPase head at the other 1 . All three SMC heterodimers interact at the hinge and ATP binding/hydrolysis occurs in two pockets formed between the heads of the two subunits. For all SMC complexes, ATP turnover is essential for cell viability and has been proposed to bring about conformational changes in the arms 13,14,15 .The ATPase activity is also key to the interaction of SMC's with DNA: Cohesin's ATPase is required for both loading and dissociation from DNA 16 , whilst condensin is dependent on its ATPase activity for translocating along DNA and forming loop structures 17,18 . The role of the Smc5/6 ATPase in DNA asso...

Efficient protein synthesis in eukaryotes requires diphthamide modification of translation elongation factor eEF2 and wobble uridine modifications of tRNAs. In higher eukaryotes, these processes are important for preventing neurological and developmental defects and cancer. In this study, we used Schizosaccharomyces pombe as a model to analyse mutants defective in eEF2 modification (dph1Δ), in tRNA modifications (elp3Δ), or both (dph3Δ) for sensitivity to cytotoxic agents and thermal stress. The dph3Δ and elp3Δ mutants were sensitive to a range of drugs and had growth defects at low temperature. dph3Δ was epistatic with dph1Δ for sensitivity to hydroxyurea and methyl methanesulfonate, and with elp3Δ for methyl methanesulfonate and growth at 16 °C. The dph1Δ and dph3Δ deletions rescued growth defects of elp3Δ in response to thiabendazole and at 37 °C. Elevated tRNALys UUU levels suppressed the elp3Δ phenotypes and some of the dph3Δ phenotypes, indicating that lack of tRNALys UUU modifications were responsible. Furthermore, we found positive genetic interactions of elp3Δ and dph3Δ with sty1Δ and atf1Δ, indicating that Elp3/Dph3-dependent tRNA modifications are important for efficient biosynthesis of key factors required for accurate responses to cytotoxic stress conditions.

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.Electronic supplementary materialThe online version of this article (doi:10.1007/s00294-017-0711-x) contains supplementary material, which is available to authorized users.

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSa (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLa (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSb (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade + reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSa-and MutLa-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2 FEN1 . Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.

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