SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint

Abstract

Cells have an intricate signaling network that deals with genomic insults (Weinert 1998; Zhou and Elledge 2000). This signaling network in response to DNA damage is composed of interacting signal transduction pathways, each consisting of sensors, transducers, and effectors. Sensors detect damaged DNA and signal to transducers. Transducers amplify and relay the signal to effectors. Effectors then execute the cellular response to elicit cell cycle checkpoint activation, DNA repair or apoptosis. Many tumor suppressor proteins are components of the DNA damage signaling network, underscoring the importance of this network to cancer development.

Proteins that serve as sensors are not well defined. Prime candidates are three groups of proteins that contain functional motifs: (1) PCNA-like proteins Rad1/Rad9/Hus1, (2) RFC-like proteins Rad17/RFC2–5, and (3) BRCT domain-containing proteins Rad9/DPB11 in Saccharomyces cerevisiae and Crb2/Rhp9/Cut5 in Schizosaccharomyces pombe (the mammalian counterparts are not known, but the breast cancer tumor suppressor protein BRCA1 and a protein called 53BP1 are candidates; Zhou and Elledge 2000). The double-stranded break (DSB) repair protein complex MRE11/RAD50/NBS1 (M/R/N) is also hypothesized to be a sensor, as it localizes to the region of DSBs in response to ionizing radiation (IR) (Maser et al. 1997; Nelms et al. 1998; Mirzoeva and Petrini 2001). The central signal transducer in response to IR is the checkpoint kinase ATM, the protein product of the gene mutated in ataxia-telangiectasia (A-T) (Shiloh and Rotman 1996). ATM is responsible for the activation of the G₁, S, and G₂/M checkpoints (Shiloh 2001). Tumor suppressor proteins p53 and CHK2 serve as effectors and are phosphorylated and activated by ATM to induce G₁ and G₂/M cell cycle arrest (Banin et al. 1998; Canman et al. 1998; Matsuoka et al. 1998).

The defective S-phase checkpoint is defined by radioresistant DNA synthesis (RDS). In S-phase checkpoint proficient cells, the rate of DNA synthesis decreases in response to IR. This decrease occurs to a lower extent in S-phase checkpoint defective cells. A-T and NBS (derived from the Nijmegen breakage syndrome) cells were first noted for this defect (Painter and Young 1980). One pathway involved in the activation of the S-phase checkpoint is ATM/CHK2/CDC25A (Falck et al. 2001). ATM activates CHK2, and CHK2 phosphorylates the cell cycle regulator CDC25A, leading to its degradation through the polyubiquitination-mediated proteolysis pathway. ATM also phosphorylates NBS1 to activate the S-phase checkpoint (Gatei et al. 2000; Lim et al. 2000; Wu et al. 2000; Zhao et al. 2000), but the downstream effectors are not known, and the relationship between the NBS1 pathway and the ATM/CHK2/CDC25A pathway is not clear. BRCA1, which is believed to function in DNA damage response and transcription regulation, is also required for activation of the S-phase checkpoint (Xu et al. 2001), and is also phosphorylated by ATM in response to IR (Cortez et al. 1999).

Structural maintenance of chromosomes (SMC) proteins are evolutionarily conserved chromosomal proteins. SMC proteins contain coiled-coil domains flanked by globular N- and C-terminal domains, and are divided in the central region by a flexible hinge domain. SMC1 and SMC3 are components of the cohesin complex, which is necessary for sister chromatid cohesion (Guacci et al. 1997; Michaelis et al. 1997; Losada et al. 1998). SMC1 and SMC3 are believed to form a heterodimer in an antiparallel fashion, in which the C-terminal coiled-coil domain of SMC1 interacts with the N-terminal coiled-coil domain of SMC3 (Strunnikov and Jessberger 1999). Cohesion between sister chromatids must be coordinated with DNA replication because cohesion is established during DNA replication (Uhlmann and Nasmyth 1998). The cohesin complex also functions in DNA repair, and is required for postreplicative DSB repair in S. cerevisiae (Sjogren and Nasmyth 2001). A mutation in one subunit of the cohesin complex in S. pombe, Rad21, renders cells sensitive to DNA damage (Birkenbihl and Subramani 1992).

We report here that SMC proteins are components of the DNA damage response network. ATM phosphorylates SMC1 in response to IR in an NBS1-dependent manner, and the phosphorylation of SMC1 is required for S-phase checkpoint activation. Our data show that SMC1 is a downstream effector in the ATM/NBS1 branch of the S-phase checkpoint pathway. We also show that NBS1 serves as an adaptor in the ATM/NBS1/SMC1 pathway. The ATM/CHK2/CDC25A pathway is intact in NBS cells. Therefore, the ATM/NBS1/SMC1 pathway defines a separate branch of the S-phase checkpoint that is distinct from the ATM/CHK2/CDC25A pathway.

Acknowledgments

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