Structural maintenance of chromosomes (SMC) proteins (SMC1, SMC3) are evolutionarily conserved chromosomal proteins that are components of the cohesin complex, necessary for sister chromatid cohesion. These proteins may also function in DNA repair. Here we report that SMC1 is a component of the DNA damage response network that functions as an effector in the ATM/NBS1-dependent S-phase checkpoint pathway. SMC1 associates with BRCA1 and is phosphorylated in response to IR in an ATM-and NBS1-dependent manner. Using mass spectrometry, we established that ATM phosphorylates S957 and S966 of SMC1 in vivo. Phosphorylation of S957 and/or S966 of SMC1 is required for activation of the S-phase checkpoint in response to IR. We also discovered that the phosphorylation of NBS1 by ATM is required for the phosphorylation of SMC1, establishing the role of NBS1 as an adaptor in the ATM/NBS1/SMC1 pathway. The ATM/CHK2/CDC25A pathway is also involved in the S-phase checkpoint activation, but this pathway is intact in NBS cells. Our results indicate that the ATM/NBS1/SMC1 pathway is a separate branch of the S-phase checkpoint pathway, distinct from the ATM/CHK2/CDC25A branch. Therefore, this work establishes the ATM/NBS1/SMC1 branch, and provides a molecular basis for the S-phase checkpoint defect in NBS cells.

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mrel1. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mrel1/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.

Several newly identified tumor suppressor genes including ATM, NBS1, BRCA1 and BRCA2 are involved in DNA double-strand break repair (DSBR) and DNA damage-induced checkpoint activation. Many of the gene products involved in checkpoint control and DSBR have been studied in great detail in yeast. In addition to evolutionarily conserved proteins such as Chk1 and Chk2, studies in mammalian cells have identified novel proteins such as p53 in executing checkpoint control. DSBR proteins including Mre11, Rad50, Rad51, Rad54, and Ku are present in yeast and in mammals. Many of the tumor suppressor gene products interact with these repair proteins as well as checkpoint regulators, thus providing a biochemical explanation for the pleiotropic phenotypes of mutant cells. This review focuses on the proteins mediating G1/S, S, and G2/M checkpoint control in mammalian cells. In addition, mammalian DSBR proteins and their activities are discussed. An intricate network among DNA damage signal transducers, cell cycle regulators and the DSBR pathways is illustrated. Mouse knockout models for genes involved in these processes have provided valuable insights into their function, establishing genomic instability as a major contributing factor in tumorigenesis.

The monocytic leukemia zinc finger protein MOZ and the related factor MORF form tetrameric complexes with ING5 (inhibitor of growth 5), EAF6 (Esa1-associated factor 6 ortholog), and the bromodomain-PHD finger protein BRPF1, -2, or -3. To gain new insights into the structure, function, and regulation of these complexes, we reconstituted them and performed various molecular analyses. We found that BRPF proteins bridge the association of MOZ and MORF with ING5 and EAF6. An N-terminal region of BRPF1 interacts with the acetyltransferases; the enhancer of polycomb (EPc) homology domain in the middle part binds to ING5 and EAF6. The association of BRPF1 with EAF6 is weak, but ING5 increases the affinity. These three proteins form a trimeric core that is conserved from Drosophila melanogaster to humans, although authentic orthologs of The gene of MOZ (monocytic leukemia zinc finger protein, also referred to as MYST3 and KAT6A), located on chromosome 8p11, was first identified as a fusion partner in chromosome translocation t(8;16)(p11;p13) (2, 52). This recurrent translocation is associated with a monocytic subtype of acute myeloid leukemia and results in the fusion of the MOZ Nterminal domain to the C-terminal part of the transcription coactivator CBP. Two other leukemia-associated chromosomal rearrangements lead to the expression of proteins fusing MOZ fragments to the CBP paralog p300 and the p300/CBP-interacting nuclear receptor coactivator TIF2 (transcription intermediary factor 2, also known as steroid receptor coactivator 2 [SRC-2] and nuclear receptor coactivator 2 [NCOA2]) (6,8,29,34). One of the resulting fusion proteins, MOZ-TIF2, is known to promote self-renewal of leukemic stem cells (17,25), suggesting that the chromosome abnormalities play a causal role in leukemogenesis. In addition, it was recently reported that MOZ is fused to NCOA3 (22), a TIF2 paralog synonymous with SRC-3 and AIB1 (amplified in breast cancer 1). MOZ is highly homologous to MORF (MOZ-related factors, also named Querkopf, MYST4, and KAT6B) (11,64). The MORF gene is rearranged in leukemia patients with t(10; 16)(q22;p13) (46) and in leiomyoma cases with t(10;17)(p11; q21) (40). The CBP gene is the fusion partner in the former translocation, while the GCN5 gene is a potential candidate in the latter translocation. All of these findings suggest that deregulated acetylation has an important role in oncogenesis. In addition, recent studies indicate that MOZ and MORF play key roles in hematopoiesis, skeletogenesis, neurogenesis, and other developmental processes (16,26,38,39,62,64). Therefore, MOZ and MORF are intimately linked to both normal development and cancer development (63,69).At the molecular level, available data suggest that this pair of paralogs functions as transcriptional coactivators with intrinsic histone acetyltransferase (HAT) activity (3,11,12,27,28,48). Both possess the MYST domain, a catalytic core conserved among members of the MYST family of acetyltransferases (2, 52). Within this family, there are five members in hu...

No abstract