Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage

Abstract

Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. Here, we show that the protein kinase, Atm, which belongs to a family of phosphatidylinositol 3-kinases that regulate cell cycle checkpoints and DNA recombination and repair, phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway. Signal transduction pathways activated by DNA damage and other cellular stresses are critical determinants of cell survival and cellular transformation. The product of the ATM gene, which is mutated in the cancer-prone disorder, Ataxia-telangiectasia (A-T), appears to be a major regulator of cellular responses to ionizing irradiation . Cells from A-T patients exhibit a variety of abnormalities, including genetic instability, radiosensitivity, cell cycle checkpoint defects in the G 1 , S, and G 2 phases of the cell cycle, and poor growth in culture. Atm is a protein kinase that is activated by ionizing irradiation (Banin et al. 1998;Canman et al. 1998) and that phosphorylates a number of different substrates following activation, including p53, Mdm2, Chk2, Nbs1, Brca1, and Rad17 (Kastan and Lim 2000; Zhou and Elledge 2000; Shiloh and Kastan 2001). The functional consequences of some of these phosphorylation events have been elucidated. For example, Atm phosphorylation of p53 (Banin et al. 1998;Canman et al. 1998), Chk2 (Matsuoka et al. 1998Ahn et al. 2000;Melchionna et al. 2000;, and Mdm2 (Maya et al. 2001) appears to contribute to the arrest of cells in G 1 following ionizing irradiation whereas phosphorylation of Nbs1, Brca1, and Rad17 is required for transient S phase (Nbs1 and Brca1) or G 2 (Brca1 and Rad17) arrests after ionizing irradiation Zhao et al. 2000;Bao et al. 2001;Xu et al. 2001). However, not all of the phenotypic abnormalities in A-T patients and cells from A-T patients can be explained by these known phosphorylation events, t...

Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation

“…Once phosphorylated, NBN promotes the ATMmediated phosphorylation of SMC1 (at residues Ser957 and Ser966) and p53 (at the Ser15 residue). Notably, SMC1 is involved in the activation of the intra-S cell cycle checkpoint and in the entry into mitosis, while p53 regulates the G1/S checkpoint (49)(50)(51). Data obtained indicate that the proper orientation and integrity of the tandem BRCT domains of NBN seems to be crucial for the activation of both ATM and its downstream effectors.…”

Interplay between ATM and ATR in the regulation of common fragile site stability

“…Recently it was shown that downregulation of the Chk1 kinase has a major effect on fragile site stability , hence the effect of ATM on the level of Chk1 phosphorylation is probably part of the mechanism by which ATM regulates fragile site expression. It is worth noting that in addition to Chk1, other shared targets of ATR and ATM (BRCA1, SMC1 and FANCD2) (Gatei et al, 2001;Kim et al, 2002;Ho et al, 2006) were also shown to regulate fragile site stability (reviewed by Arlt et al, 2006). Further studies are required to investigate the regulation of these proteins by ATR and ATM, under conditions that induce fragile site expression.…”

Activation and regulation of ATM kinase activity in response to DNA double-strand breaks

“…(b) Nbs1 is a 90 kDa protein, consisting of 754 amino acids. The phosphorylation sites (P) show the serine residues that are phosphorylated by ATM (Gatei et al, 2000;Lim et al, 2000;Stewart et al, 2001;Kim et al, 2002;Lee et al, 2003b). The domains within Nbs1 include the forkhead-associated (FHA) domain (Featherstone and Jackson, 1998), the breast cancer C-terminal (BRCT) domains (Becker et al, 2006), the Mre11-binding domain (MBD) (Desai-Mehta et al, 2001) and the ATM-interacting domain (Falck et al, 2005).…”

“…Nbs1 is phosphorylated in an ATM-dependent manner after DNA damage on residues Ser278, Ser343 and Ser615 (Gatei et al, 2000;Lim et al, 2000;Stewart et al, 2001;Kim et al, 2002;Lee et al, 2003b). Several studies showed that the expression of S343A mutant Nbs1 in NBS cells resulted in partial defects in IR-induced S-phase checkpoint activation, radiosensitivity and partial defects in ATM-dependent phosphorylation events including Chk2, SMC1 and FANCD2 (Lim et al, 2000;Buscemi et al, 2001;Nakanishi et al, 2002;Yazdi et al, 2002).…”