Scientific Report 2006

Molecular Biology

DNA Repair and the Maintenance of Genomic Stability

M.N. Boddy, S. Pebernard, J. Prudden

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 studies 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. We expressed and purified individual components of the complex and determined the architecture of the holocomplex (Fig. 1). 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. 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.

Fig. 1. Architecture and function of the Smc5-Smc6 holocomplex.

We have carried out detailed genetic and biochemical analyses of the Nse5-Nse6 heterodimer. Nse5 and Nse6 are not essential for growth; however, cells lacking either protein have high levels of spontaneous genome damage and are hypersensitive to ultraviolet light and other genotoxic agents. An important discovery is that Nse5-Nse6 prevents the deleterious engagement of an ordinarily beneficial DNA repair pathway called homologous recombination. Our studies indicate that Nse5-Nse6, and by extension the Smc5-Smc6 complex, acts either to prevent initiation of homologous recombination or to separate physically linked chromosomes that arise late in this process (Fig. 1B). Abrogating homologous recombination by deleting a pivotal factor required for the process (called Rad51) reduces the sensitivity of Nse5-Nse6 mutant cells to DNA damage. The spontaneous DNA damage observed in Nse5-Nse6 mutant cells is due to the attempted separation of chromosomes into daughter cells while the chromosomes are still physically linked. Such defective chromosome separation in humans could result in cancer and other diseases.

Publications

Pebernard, S., Wohlschlegel, J., McDonald, W.H., Yates, J.R. III, Boddy, M.N. The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex [published correction appears in Mol. Cell. Biol. 26:3336, 2006]. Mol. Cell. Biol. 26:1617, 2006.

Raffa, G.D., Wohlschlegel, J., Yates, J.R. III, Boddy, M.N. SUMO-binding motifs mediate the Rad60-dependent response to replicative stress and self-association. J. Biol. Chem. 281:27973, 2006.