ATR is a key regulator of checkpoint responses to incompletely replicated and damaged DNA, but the mechanisms underlying control of its kinase activity are unknown. TopBP1, the vertebrate homolog of yeast Cut5/Dbp11, has dual roles in initiation of DNA replication and regulation of checkpoint responses. We show that recombinant TopBP1 induces a large increase in the kinase activity of both Xenopus and human ATR. The ATR-activating domain resides in a conserved segment of TopBP1 that is distinct from its numerous BRCT repeats. The isolated ATR-activating domain from TopBP1 induces ectopic activation of ATR-dependent signaling in both Xenopus egg extracts and human cells. Furthermore, Xenopus egg extracts containing a version of TopBP1 with an inactivating point mutation in the ATR-activating domain are defective in checkpoint regulation. These studies establish that activation of ATR by TopBP1 is a crucial step in the initiation of ATR-dependent signaling processes.

The molecular mechanisms underlying angioimmunoblastic T cell lymphoma (AITL), a common type of mature T cell lymphoma of poor prognosis, are largely unknown. Here we report a frequent somatic mutation in RHOA (encoding p.Gly17Val) using exome and transcriptome sequencing of samples from individuals with AITL. Further examination of the RHOA mutation encoding p.Gly17Val in 239 lymphoma samples showed that the mutation was specific to T cell lymphoma and was absent from B cell lymphoma. We demonstrate that the RHOA mutation encoding p.Gly17Val, which was found in 53.3% (24 of 45) of the AITL cases examined, is oncogenic in nature using multiple molecular assays. Molecular modeling and docking simulations provided a structural basis for the loss of GTPase activity in the RHOA Gly17Val mutant. Our experimental data and modeling results suggest that the RHOA mutation encoding p.Gly17Val is a driver mutation in AITL. On the basis of these data and through integrated pathway analysis, we build a comprehensive signaling network for AITL oncogenesis.

The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. We show that, during this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response.

Extranodal NK/T-cell lymphoma nasal type (ENKL) is a rare type of non-Hodgkin lymphoma that more frequently occurs in East Asia and Latin America. Even though its molecular background has been discussed in the last few years, the current knowledge does not explain the disease pathogenesis in most cases of ENKL. Here, we performed multiple types of next-generation sequencing on 34 ENKL samples, including wholeexome sequencing (9 cancer tissues and 4 cancer cell lines), targeted sequencing (21 cancer tissues), and RNA sequencing (3 cancer tissues and 4 cancer cell lines). Mutations were found most frequently in 3 genes, STAT3, BCOR, and MLL2 (which were present in 9, 7, and 6 cancer samples, respectively), whereas there were only 2 cases of JAK3 mutation. In total, JAK/STAT pathway-and histone modification-related genes accounted for 55.9% and 38.2% of cancer samples, respectively, and their involvement in ENKL pathogenesis was also supported by gene expression analysis. In addition, we provided 177 genes upregulated only in cancer tissues, which appear to be linked with angiocentric and angiodestructive growth of ENKL. In this study, we propose several novel driver genes of ENKL, and show that these genes and their functional groups may be future therapeutic targets of this disease.

Five-year survival of patients transplanted for HCC is excellent, with a steady improvement in survival over the past decade. It is possible that the published criteria for patient selection may have contributed to the better outcome.

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ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not to DNA replication stress. TopBP1 has been identified as a direct activator of ATR. We show that ATM regulates Xenopus TopBP1 by phosphorylating Ser-1131 and thereby strongly enhancing association of TopBP1 with ATR. Xenopus egg extracts containing a mutant of TopBP1 that cannot be phosphorylated on Ser-1131 are defective in the ATR-dependent phosphorylation of Chk1 in response to DSBs but not to DNA replication stress. Thus, TopBP1 is critical for the ATM-dependent activation of ATR following production of DSBs in the genome.In eukaryotic cells, a variety of checkpoint control mechanisms help to maintain genomic integrity (1-3). Elements of these regulatory systems scrutinize the genome for the existence of damaged or incompletely replicated DNA. ATM 2 (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related), which belong to the phosphoinositide kinase-related family of protein kinases, are key upstream players in checkpoint pathways (1). ATM responds principally to the occurrence of double-stranded DNA breaks (DSBs) in the genome. By contrast, ATR plays a distinct role in the detection of stalled DNA replication forks, but it also participates in DNA damage responses (1,4,5). ATR possesses a binding partner called ATRIP that is essential for checkpoint regulatory functions (6). It has been established that activation of Chk1 in response to DSBs, but not to DNA replication stress, also depends upon upstream regulation by ATM (7-9). This observation implied that certain DNA structures, but not others, rely on ATM to elicit the ATR-dependent phosphorylation of Chk1. In more recent studies, TopBP1 has been identified as a direct activator of the ATR-ATRIP complex (10). Here we demonstrate that TopBP1 has a direct and essential role in the pathway that connects ATM to ATR specifically in response to the occurrence of DSBs in the genome. EXPERIMENTAL PROCEDURESXenopus Egg Extracts-Xenopus egg extracts were prepared as described (11). Extracts were treated with 50 g/ml (dA) 70 ⅐(dT) 70 to induce checkpoint responses (12). To prepare extracts containing chromatin with double-stranded DNA breaks or DNA replication blocks, demembranated sperm nuclei (1000 -3000/ l) were incubated in extracts containing 0.05 unit/ l EcoRI or 100 g/ml aphidicolin, respectively. For preparation of nuclear fractions, egg extracts (50 l) containing 3000 sperm nuclei/ l were incubated under the indicated conditions. Nuclear fractions were prepared as described previously (13).Antibodies-For production of anti-P-Ser-1131 antibodies to phosphorylated XtopBP1, the peptide CLNTEPSQNEQI (containing residues 1126 -1136 of XtopBP1) was synthesized with a phosphate on Ser-1131 and an extra cysteine residue for conjugation to keyhole limpet hemocyanin. Anti-phosphopeptide antibodies were prepared as described (11). Other antibodies were described previously (10,14,15).Immunoprecipitation a...

Type 1 and CAD are both independent predictors of poor outcome after liver transplantation. Liver transplant recipients with type 1 or CAD have approximately 40% lower 5-year survival compared with patients without DM or CAD.