The role of the human SMC5/6 complex in genome stability

The Smc5/6 complex is a member of the SMC family of protein complexes, comprising cohesin, condensin and Smc5/6. However, in comparison to cohesin and condensin, the cellular role of the Smc5/6 complex is much less understood. Smc5/6 in essential in yeasts and its absence in mice is embryonically lethal. Studies in fission and budding yeast have reported roles for Smc5/6 in homologous recombination repair, and the maintenance of replication fork stability. Recent studies in human cells identified two different syndromes resulting from mutations in distinct SMC5/6 subunits, NSMCE2 and NSMCE3. Both syndromes are characterised by chromosomal instability and failure to recover from replication stress. To further examine the requirements for human SMC5/6, we took advantage of a T-REx Flp-In cell line, which allowed for an inducible downregulation of a specific gene of interest. The endogenous SMC5/6 subunit, NSMCE4a, was first complemented with a regulatable NSMCE4a-Strep-6xHis cDNA, and subsequently knocked out. Additional removal of the ectopic NSMCE4a in the knock out cell line demonstrated that NSMCE4a is essential in human cells. We took advantage of a bottom up proteomics approach to purify the SMC5/6 complex using NSMCE4a-Strep-6xHis and search for its interactors during an unperturbed cell cycle, upon replication fork stalling and after fork collapse. In parallel, we found that downregulation of ectopic NSMCE4a destabilises the SMC5/6 complex in T-REx Flp-In 293 cells, similarly to the NSMCE3 patient cells. SMC5/6 destabilisation results in aneuploidy, genome instability, an increase in DNA damage markers and replication stress. The cells are capable of homologous recombination as evidenced by Rad51 foci and display elevated non-allelic homologous recombination. Interestingly, we found that the depletion of SMC5/6 by NSMCE4a shut off leads to strong sensitivity to PARP inhibition. Our data suggest that this is due to a failure to protect replication fork and/or misregulation of homologous recombination.