Scientific Report 2005

Molecular Biology

DNA Repair and the Maintenance of Genomic Stability

M.N. Boddy, Y. Pavlova, S. Pebenard, G. Raffa

DNA repair pathways have evolved to protect the genome from ever-present genotoxic agents. Highlighting the importance of the pathways, defects in DNA repair mechanisms strongly predispose the host to cancer and to neurologic and developmental disorders. The DNA repair systems we study in fission yeast are evolutionarily conserved, and therefore our investigations provide a valuable framework for understanding genome maintenance in human cells.

Although many DNA repair mechanisms have been described, information on how they are coordinated with necessary changes in chromatin structure is limited. We are studying the essential structural maintenance of chromosomes (SMC) complex Smc5-Smc6. The molecular functions of Smc5-Smc6 are unknown, but the complex is related to the SMC complexes that hold replicated sister chromatids together (cohesin) and condense chromatin before its segregation at mitosis (condensin).

In collaboration with J.R. Yates, Department of Cell Biology, we purified the Smc5-Smc6 complex and determined the identity of the core components. The holocomplex consists of the Smc5-Smc6 heterodimer and 6 additional non-SMC elements, Nse1–Nse6 (Fig. 1).

Fig. 1. Architecture of the Smc5-Smc6 holocomplex. Nse1, Nse3, and Nse4 form a stable heterotrimer that then associates with Smc5. Nse2 interacts directly with Smc5 in the absence of the other Nse proteins. Smc6 interacts directly with Smc5 but none of the other components. Nse5 and Nse6 form a stable heterodimer that also binds directly to Smc5. Double-headed arrows indicate interactions between subcomplexes. Nse5-Nse6 may recruit the holocomplex to stalled replication forks and certain DNA damage sites (black oval on leading-strand template of replication fork).

We expressed and purified individual components of the complex and determined the architecture of the holocomplex. Nse1–Nse4 are essential for growth, and hypomorphic mutants of these proteins cause cellular sensitivity to genotoxic agents such as ultraviolet light and x-rays. Nse5 and Nse6 are nonessential, but cells lacking either protein also are hypersensitive to DNA-damaging agents. Notably, Nse1 and Nse2 contain certain zinc finger domains that implicate these 2 elements in the modification of target proteins with ubiquitin and the small ubiquitin-like protein SUMO. Such protein modifications play roles in DNA repair and chromatin remodeling.

Our genetic analyses support a role for the Smc5-Smc6 complex in stabilizing replication forks that have stalled at sites of DNA damage. We have also identified a critical role for the Smc5-Smc6 complex in meiosis, the process that generates gametes for reproduction and genetic diversity. A critical feature of meiosis is the programmed formation of DNA double-strand breaks followed by repair of the breaks via homologous recombination. We found that the Smc5-Smc6 complex functions in the correct repair of the breaks and that mutants of Smc5-Smc6 do not segregate homologous chromosomes at the first meiotic division.

Finally, we identified a physical interaction between the Smc5-Smc6 complex and Rad60, an essential DNA repair factor required for the homologous recombination repair of DNA. Rad60 is regulated by the replication checkpoint, and thus we can study the important but poorly defined interface between DNA repair and cell-cycle checkpoints.

Publications

Kai, M., Boddy, M.N., Russell, P., Wang, T.S. Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. Genes Dev. 19:919, 2005.

Pebernard, S., McDonald, W.H., Pavlova, Y., Yates, J.R., III, Boddy, M.N. Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis. Mol. Biol. Cell 15:4866, 2004.