Condensin recruitment to chromatin is inhibited by Chk2 kinase in response to DNA damage

The DNA damage checkpoint, when activated in response to genotoxic damage during S phase, arrests cells in G2 phase of the cell cycle. ATM, ATR, Chk1 and Chk2 kinases are the main effectors of this checkpoint pathway. The checkpoint kinases prevent the onset of mitosis by eliciting well characterized inhibitory phosphorylation of Cdk1. Since Cdk1 is required for the recruitment of condensin, it is thought that upon DNA damage the checkpoint also indirectly blocks chromosome condensation via Cdk1 inhibition. Here we report that the G2 damage checkpoint prevents stable recruitment of the chromosome-packaging-machinery components condensin complex I and II onto the chromatin even in the presence of an active Cdk1. DNA damage-induced inhibition of condensin subunit recruitment is mediated specifically by the Chk2 kinase, implying that the condensin complexes are targeted by the checkpoint in response to DNA damage, independently of Cdk1 inactivation. Thus, the G2 checkpoint directly prevents stable recruitment of condensin complexes to actively prevent chromosome compaction during G2 arrest, presumably to ensure efficient repair of the genomic damage.     

Widdrol activates DNA damage checkpoint through the signaling Chk2–p53–Cdc25A–p21–MCM4 pathway in HT29 cells

Widdrol is an odorant compound isolated from Juniperus chinensis. We previously reported that widdrol induces Gap 1 (G1) phase cell cycle arrest and leads to apoptosis in human colon adenocarcinoma HT29 cells. It was also reported that this cell cycle arrest is associated with the induction of checkpoint kinase 2 (Chk2), p53 phosphorylation and cyclin dependent kinase (Cdk) inhibitor p21 expression. In this paper, we investigated the molecular mechanisms of widdrol on the activation of G1 DNA damage checkpoint at early phase when DNA damages occurred in HT29 cells. First of all, we examined that widdrol breaks DNA directly or not. As the results of DNA electrophoresis and formation of phosphorylated histone H2AX (γH2AX) foci in HT29 cells, widdrol generates DNA double-strand breaks directly within 0.5?h both in vitro and in vivo. Based on this result, the change of proteins related in checkpoint pathway was examined over a time course of 0.5-24?h. Treatment of HT29 cells with widdrol elicits the following: (1) phosphorylation of Chk2 and p53, (2) reduction of cell division cycle 25A (Cdc25A) expression, (3) increase of Cdk inhibitor p21 expression, and (4) decrease of the levels of Cdk2 and cyclin E expression in a time-dependent manner. Moreover, only the expression level of mini-chromosome maintenance 4 (MCM4) protein, a subunit of the eukaryotic DNA replicative helicase, is rapidly down-regulated in HT29 cells treated with widdrol over the same time course, but those of the other MCM proteins are unchanged. Overall, our results indicated that widdrol breaks DNA directly in HT29 cells, and this DNA damage results in checkpoint activation via Chk2-p53-Cdc25A-p21-MCM4 pathway and finally cells go to G1-phase cell cycle arrest and apoptosis.     

A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

It has been reported previously that both Cdk1 and Cdk2 phosphorylate Chk1 in a cell-cycle dependent manner. Cdk-mediated phosphorylation is required for efficient activation of Chk1 and checkpoint proficiency in response to DNA damage. Here, we demonstrate that Cdk-mediated phosphorylation is also required for replication stress induced Chk1 activation and S/M checkpoint proficiency. Re-introduction of Chk1 mutant (S286A/S301A) into Chk1 deficient cells is capable of restraining mitosis in cells with completely unreplicated DNA, but the mitotic delay at later stage of the cell cycle is largely impaired. The mutation strongly attenuates aphidicolin induced Chk1 activation without altering the S-phase dependent Chk1 activation. These data indicate that Cdk-mediated phosphorytion is required for efficient Chk1 activation and multiple checkpoint proficiency.     

Mitotic DNA damage response: Polo-like Kinase-1 is dephosphorylated through ATM-Chk1 pathway

DNA damage during the cell division cycle can activate ATM/ATR and their downstream kinases that are involved in the checkpoint pathway, and cell growth is halted until damage is repaired. As a result of DNA damage induced in mitotic cells by doxorubicin treatment, cells accumulate in a G2-like phase, not in mitosis. Under these conditions, two mitosis-specific kinases, Cdk1 and Plk1, are inhibited by inhibitory phosphorylation and dephosphorylation, respectively. G2-specific phosphorylation of Cdc25 was increased during incubation after mitotic DNA damage. Inhibition of Plk1 through dephosphorylation was dependent on ATM/Chk1 activity. Depleted expression of ATM and Chk1 was achieved using small hairpin RNA (shRNA) plasmid constructs. In this condition, damaged mitotic cells did not accumulated in a G2-like stage, and entered into G1 phase without delay. Protein phosphatase 2A was responsible for dephosphorylation of mitotic Plk1 in response to DNA damage. In knockdown of PP2A catalytic subunits, Plk1 was not dephosphorylated, but rather degraded in response to DNA damage, and cells did not accumulate in G2-like phase. The effect of ATM/Chk1 inhibition was counteracted by overexpression of PP2A, indicated that PP2A may function as a downstream target of ATM/Chk1 at a mitotic DNA damage checkpoint, or may have a dominant effect on ATM/Chk1 function at this checkpoint. Finally, we have shown that negative regulation of Plk1 by dephosphorylation is important to cell accumulation in G2-like phase at the mitotic DNA damage checkpoint, and that this ATM/Chk1/PP2A pathway independent on p53 is a novel mechanism of cellular response to mitotic DNA damage.     

Phosphorylation of Minichromosome Maintenance Protein 7 (MCM7) by Cyclin/Cyclin-dependent Kinase Affects Its Function in Cell Cycle Regulation

MCM7 is one of the subunits of the MCM2–7 complex that plays a critical role in DNA replication initiation and cell proliferation of eukaryotic cells. After forming the pre-replication complex (pre-RC) with other components, the MCM2–7 complex is activated by DDK/cyclin-dependent kinase to initiate DNA replication. Each subunit of the MCM2–7 complex functions differently under regulation of various kinases on the specific site, which needs to be investigated in detail. In this study, we demonstrated that MCM7 is a substrate of cyclin E/Cdk2 and can be phosphorylated on Ser-121. We found that the distribution of MCM7-S121A is different from wild-type MCM7 and that the MCM7-S121A mutant is much less efficient to form a pre-RC complex with MCM3/MCM5/cdc45 compared with wild-type MCM7. By using the Tet-On inducible HeLa cell line, we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry, suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. Further analysis indicates that the Chk1 pathway is activated in MCM7-overexpressed cells in a p53-dependent manner. We performed experiments with the human normal cell line HL-7702 and also observed that overexpression of MCM7 can cause S phase block through checkpoint activation. In addition, we found that MCM7 could also be phosphorylated by cyclin B/Cdk1 on Ser-121 both in vitro and in vivo. Furthermore, overexpression of MCM7-S121A causes an obvious M phase exit delay, which suggests that phosphorylation of MCM7 on Ser-121 in M phase is very important for a proper mitotic exit. These data suggest that the phosphorylation of MCM7 on Ser-121 by cyclin/Cdks is involved in preventing DNA rereplication as well as in regulation of the mitotic exit.     

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.     

p53 Regulates Cellular Responses to Environmental Carcinogen Benzo[a]pyrene-7,8-diol-9,10-epoxide in Human Lung Cancer Cells

The p53 tumor suppressor is a mutational target of environmental carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). We now demonstrate that p53 plays an important role in regulation of cellular responses to BPDE. Exposure of p53-null H1299 human lung cancer cells to BPDE resulted in S and G2 phase cell cycle arrest, but not mitotic block, which correlated with induction of cyclin B1 protein expression, down-modulation of cell division cycle 25C (Cdc25C) and Cdc25B protein levels, and hyperphosphorylation of Cdc25C (S216), cyclin-dependent kinase 1 (Cdk1; Y15), checkpoint kinase 1 (Chk1; S317 and S345) and Chk2 (T68). The BPDE-induced S phase block, but not the G2/M phase arrest, was significantly attenuated by knockdown of Chk1 protein level. The BPDE-mediated accumulation of sub-diploid fraction (apoptotic cells) was significantly decreased in H1299 cells transiently transfected with both Chk1 and Chk2 specific siRNAs. The H460 human lung cancer cell line (wild-type p53) was relatively more sensitive to BPDE-mediated growth inhibition and enrichment of sub-diploid apoptotic fraction compared with H1299 cells. The BPDE exposure failed to activate either S or G2 phase checkpoint in H460 cells. Instead, the BPDE-treated H460 cells exhibited a nearly 8-fold increase in sub-diploid apoptotic cells that was accompanied by phosphorylation of p53 at multiple sites. Knockdown of p53 protein level in H460 cells attenuated BPDE-induced apoptosis but enforced activation of S and G2 phase checkpoints. In conclusion, the present study points towards an important role of p53 in regulation of cellular responses to BPDE in human lung cancer cells.     

Increases in mitochondrial DNA content and 4977-bp deletion upon ATM/Chk2 checkpoint activation in HeLa cells

Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.     

The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest

Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G?/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.     

Onset of the DNA replication checkpoint in the early Drosophila embryo

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.     

Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest

Cyclin E is a regulator of cyclin-dependent protein kinases (Cdks) and is involved in mediating the cell cycle transition from G₁ to S phase. Here, we describe a novel function for cyclin E in the long term maintenance of checkpoint arrest in response to replication barriers. Exposure of cells to mitomycin C or UV irradiation, but not ionizing radiation, induces stabilization of cyclin E. Stabilization of cyclin E reduces the activity of Cdk2-cyclin A, resulting in a slowing of S phase progression and arrest. In addition, cyclin E is shown to be required for stabilization of Cdc6, which is required for activation of Chk1 and the replication checkpoint pathway. Furthermore, the stabilization of cyclin E in response to replication fork barriers depends on ATR, but not Nbs1 or Chk1. These results indicate that in addition to its well studied role in promoting cell cycle progression, cyclin E also has a role in regulating cell cycle arrest in response to DNA damage.     

Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle

MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex.     

Tight Chk1 Levels Control Replication Cluster Activation in Xenopus

DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.     

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the beta-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the beta-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.     

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ～10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.     

The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.     

p21 Inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint

Cdk1 was proposed to compensate for the loss of Cdk2. Here we present evidence that this is possible due to premature translocation of Cdk1 from the cytoplasm to the nucleus in the absence of Cdk2. We also investigated the consequence of loss of Cdk2 on the maintenance of the G1/S DNA damage checkpoint. Cdk2(-/-) mouse embryonic fibroblasts in vitro as well as regenerating liver cells after partial hepatectomy (PH) in Cdk2(-/-) mice, arrest promptly at the G1/S checkpoint in response to gamma-irradiation due to activation of p53 and p21 inhibiting Cdk1. Furthermore re-entry into S phase after irradiation was delayed in Cdk2(-/-) cells due to prolonged and impaired DNA repair activity. In addition, Cdk2(-/-) mice were more sensitive to lethal irradiation compared to wild-type and displayed delayed resumption of DNA replication in regenerating liver cells. Our results suggest that the G1/S DNA damage checkpoint is intact in the absence of Cdk2, but Cdk2 is important for proper repair of the damaged DNA.     

The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage.     

Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form γ-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.