Coordination of DNA Damage Responses via the Smc5/Smc6 Complex

Abstract

The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.Cell cycle checkpoints are surveillance mechanisms that monitor the order and fidelity of cell cycle events. Among these are the DNA integrity checkpoints, which monitor DNA replication and DNA damage. Ongoing or stalled replication activates a checkpoint that prevents initiation of mitosis, ensuring the interdependence of the S phase and mitosis to maintain ploidy. There is considerable overlap between this checkpoint and that which monitors DNA damage during the S and G 2 phases of the cell cycle, at which time there is the added challenge of coordinating cell cycle progression with successful DNA repair (8).The biology of the G 2 DNA damage checkpoint in the fission yeast Schizosaccharomyces pombe has been extensively studied (40). This checkpoint arrests the cell cycle through maintenance of the inhibitory tyrosine-15 phosphorylation of Cdc2, which is achieved through Chk1-dependent signaling to the Cdc25 phosphatase and the Wee1 kinase that regulate Cdc2 (15,39,41,42). The earliest molecular marker of the G 2 DNA damage response is Rad3-dependent phosphorylation of its binding partner Rad26, the homolog of human ATR and ATRIP (9, 12). Phosphorylation of Chk1 following DNA damage is mediated by Rad3 and requires several other proteins encoded by the checkpoint rad genes, rad1, hus1, rad9, and rad17, together with the BRCT domain protein, Crb2 (29,43,56). These proteins, and the Rad3 and Rad26 homologs, have also been shown to independently localize to the sites of DNA damage in both Saccharomyces cerevisiae and human cells (23,35,58). Although Rad3/ATR activation is an early checkpoint response to DNA damage, the actual sensors of DNA lesions are unknown and may differ depending on the specific nature of the lesion (3, 59).Exper...

Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

“…The majority of aberrations observed have DNA stretched out along the axis of the mitotic spindle. This phenotype matches well that recently described for the loss of function of two other components of the Smc5ϩ6 complex, Nse1 and Smc6 Harvey et al, 2004). The nse3-1 phenotype could be consistent with a failure to fully condense chromosomes or, a physical impediment to their segregation such as sister chromatid catenation and unresolved recombination structures.…”

“…SMC heterodimers need to associate with specific non-SMC family subunits to be functional. Non-SMC subunits of the cohesin, condensin and Smc5ϩ6 complexes have been isolated and characterized (Fujioka et al, 2002;Hirano, 2002;Jessberger, 2003;McDonald et al, 2003;Harvey et al, 2004). Mutants of these non-SMC subunits display phenotypes very similar to those of the SMC mutants, consistent with their interdependent functions.…”

“…The hinge allows SMC proteins to fold back on themselves, forming an intramolecular coiled-coil and creating an ATPase by juxtaposing the Walker A and B domains (Haering et al, 2002). SMC proteins form stable heterodimers, most likely through interactions mediated by their hinge regions (Haering et al, 2002).Hypomorphic mutations of proteins in the SMC complexes render cells hypersensitive to genotoxic stress (Birkenbihl and Subramani, 1992;Lehmann et al, 1995;Sjogren and Nasmyth, 2001;Aono et al, 2002;Fujioka et al, 2002;Kim et al, 2002b;Yazdi et al, 2002;McDonald et al, 2003;Harvey et al, 2004). Cohesin may facilitate the homologous recombination repair of DSBs by holding sister-chromatids in proximity, promoting identification of an intact homologous duplex.…”

“…Like cohesin and condensin, the Smc5ϩ6 complex is essential for viability and seems to control chromatin structure (Lehmann et al, 1995;Harvey et al, 2004). Studies on mutants of the complex in fission yeast have demonstrated that it has a role in the HR-based repair of DNA damage and that it may also influence checkpoint maintenance (Lehmann et al, 1995;Verkade et al, 1999;McDonald et al, 2003;Harvey et al, 2004). SMC heterodimers need to associate with specific non-SMC family subunits to be functional.…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…SMC protein functions have been strongly implicated in DNA damage responses and DNA repair processes. It has been shown that cohesin, SMC5/SMC6 and Rad50 (another SMC protein) all localize to sites of DSBs, directing homologous recombination DNA repair and inducing S phase checkpoint (Lehmann, 2005;Kim et al, 2002;Yazdi et al, 2002;Kitagawa et al, 2004;Harvey et al, 2004).…”