Disruption of a ∼23-24 nucleotide small RNA pathway elevates DNA damage responses in Tetrahymena thermophila.

Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, Tetrahymena thermophila, the cellular purpose of RNAi pathways that generate ∼23-24 nucleotide (nt) small (s)RNAs has remained unknown. Here, we investigated the phenotypic and gene expression impacts on vegetatively growing cells when genes involved in ∼23-24 nt sRNA biogenesis are disrupted. We observed slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. Together, our findings suggest that an evolutionarily conserved role for RNAi pathways in maintaining genome integrity may be extended even to the early branching eukaryotic lineage that gave rise to Tetrahymena thermophila.

Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo.

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 single-stranded DNA (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.

Specialized interfaces of Smc5/6 control hinge stability and DNA association.

The Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6 are involved in the organization of higher-order chromosome structure-which is essential for accurate chromosome duplication and segregation. Each complex is scaffolded by a specific SMC protein dimer (heterodimer in eukaryotes) held together via their hinge domains. Here we show that the Smc5/6-hinge, like those of cohesin and condensin, also forms a toroidal structure but with distinctive subunit interfaces absent from the other SMC complexes; an unusual 'molecular latch' and a functional 'hub'. Defined mutations in these interfaces cause severe phenotypic effects with sensitivity to DNA-damaging agents in fission yeast and reduced viability in human cells. We show that the Smc5/6-hinge complex binds preferentially to ssDNA and that this interaction is affected by both 'latch' and 'hub' mutations, suggesting a key role for these unique features in controlling DNA association by the Smc5/6 complex.