Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.

Somatic mammalian cells possess well-established S-phase programs with specific regions of the genome replicated at precise times. The ATR–Chk1 pathway plays a central role in these programs, but the mechanism for how Chk1 regulates origin firing remains unknown. We demonstrate here the essential role of cyclin A2–Cdk1 in the regulation of late origin firing. Activity of cyclin A2–Cdk1 was hardly detected at the onset of S phase, but it was obvious at middle to late S phase under unperturbed condition. Chk1 depletion resulted in increased expression of Cdc25A, subsequent hyperactivation of cyclin A2–Cdk1, and abnormal replication at early S phase. Hence, the ectopic expression of cyclin A2–Cdk1AF (constitutively active mutant) fusion constructs resulted in abnormal origin firing, causing the premature appearance of DNA replication at late origins at early S phase. Intriguingly, inactivation of Cdk1 in temperature-sensitive Cdk1 mutant cell lines (FT210) resulted in a prolonged S phase and inefficient activation of late origin firing even at late S phase. Our results thus suggest that cyclin A2–Cdk1 is a key regulator of S-phase programs.

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (␥H2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by ␥H2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.Initiation of mitosis in mammals is triggered by the abrupt activation of cyclin B-Cdc2. Activation of Cdc2 is regulated by a complex process that includes the binding to its regulatory subunit, cyclin B, whose level rises at late S phase, and peaks in mitosis (1-4). Just before mitosis, most cyclin B-Cdc2 complexes exist in an inactive state because of the inhibitory phosphorylation of Cdc2 on Thr-14 and Tyr-15, whose phosphorylation is catalyzed by Wee1 and Myt1, and whose dephosphorylation is catalyzed by Cdc25 phosphatases.Mitotic catastrophe was first identified in certain mutant fission yeast strains as a lethal phenotype characterized by gross abnormalities in chromosome segregation during mitosis (5, 6). A similar lethal phenotype was also observed in mammalian cells and found to result from premature mitosis (7, 8) or a failure to undergo complete mitosis (9, 10). However, there is still no broadly accepted definition of the term "mitotic catastrophe," presumably because the major processes involved have not been described in molecular and genetic terms. For example, some researchers have argued that mitotic catastrophe is fundamentally different from apoptosis (11) because overexpression of anti-apoptotic genes including Bcl-2 and MDR1 can actually enhance the frequency of catastrophic mitosis (12, 13). In contrast, a recent study demonstrated that mitotic catastrophe induced by DNA damage is dependent on caspase activation, suggesting that it constitutes a special case of apoptosis (14,15).In response to blocks to DNA replication, eukaryotes activate checkpoint pathways that prevent genomic instability. In yeast models, spontaneous chromosomal breaks and gross chromosomal rearrangements has been detected in mutant strains that cannot respond to stalled or incomplete DNA replicat...

A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.Supplemental material is available at http://www.genesdev.org.

Effects of rice bran agglutinin (RBA) on human monoblastic leukemia U937 cells were examined in comparison with those of wheat germ agglutinin (WGA) and Viscum album agglutinin (VAA). These lectins inhibit cell growth, and several lines of evidence indicate that the growth inhibition is caused by the induction of apoptosis. We observed that RBA induces chromatin condensation, externalization of membrane phosphatidylserine, and DNA ladder formation, features of apoptosis. DNA ladder formation was inhibited by a general inhibitor against caspases, which are known to play essential roles in apoptosis. Flow cytometric analysis revealed that RBA and WGA cause G2/M phase cell cycle arrest with increased expression of Waf1/p21, while cell cycle arrest was not observed for VAA. These data indicate that RBA induces apoptosis associated with cell cycle arrest in U937 cells, and suggest that the induction mechanism for RBA is similar to that for WGA, but different from that for VAA.

The kinase Chk2 and tumor suppressor p53 participate in an ill defined regulatory interaction in mammalian cells. The abundance of Chk2 mRNA and protein has now been shown to be decreased by the induction of p53 in Saos2 cells. Ionizing radiation also triggered the phosphorylation and subsequent down-regulation of Chk2 in human colorectal HCT116 (p53 ؉/؉ ) cancer cells; irradiation of its isogenic mutant HCT116 (p53 ؊/؊ ) cells, which lack functional p53, induced Chk2 phosphorylation but not its down-regulation. In addition, HCT116 (p53 ؉/؉ ) cells constitutively expressing a dominant negative p53 (V143A) failed to suppress Chk2 expression after irradiation. Reporter gene assays in HCT116 (p53 ؉/؉ ) cells revealed that wild-type p53 repressed, whereas a dominant negative p53 mutant increased, the activity of the human Chk2 gene promoter. Mutational analysis showed that a CCAAT box located between nucleotides ؊152 and ؊138 of the promoter was responsible for its negative regulation by p53. Electrophoretic mobility shift assays demonstrated that the transcription factor NF-Y binds to this CCAAT sequence. A dominant negative mutant of NF-YA abolished the effect of p53 on Chk2 promoter activity. These results suggest that p53 negatively regulates Chk2 gene transcription through modulation of NF-Y function and that this regulation may be important for reentry of cells into the cell cycle after DNA damage is repaired.

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