细胞DNA损伤检控点

细胞周期检控点是维持细胞基因组稳定性的一个重要机制,主要包括。DNA损伤检控点、DNA复制检控点和纺锤体组装检控点。其中DNA损伤检控点能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,为修复损伤提供足够的时间,以保证细胞遗传的稳定性。有关DNA损伤检控点的研究近年来已经取得了突破性进展,现简要介绍近年来在DNA损伤检控点研究中的一些新进展。     

Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability

DNA damage response mechanisms encompass pathways of DNA repair, cell cycle checkpoint arrest and apoptosis. Together, these mechanisms function to maintain genomic stability in the face of exogenous and endogenous DNA damage. ATM is activated in response to double strand breaks and initiates cell cycle checkpoint arrest. Recent studies in human fibroblasts have shown that ATM also regulates a mechanism of end-processing that is required for a component of double strand break repair. Human fibroblasts rarely undergo apoptosis after ionising radiation and, therefore, apoptosis is not considered in our review. The dual function of ATM raises the question as to how the two processes, DNA repair and checkpoint arrest, interplay to maintain genomic stability. In this review, we consider the impact of ATM's repair and checkpoint functions to the maintenance of genomic stability following irradiation in G2. We discuss evidence that ATM's repair function plays little role in the maintenance of genomic stability following exposure to ionising radiation. ATM's checkpoint function has a bigger impact on genomic stability but strikingly the two damage response pathways co-operate in a more than additive manner. In contrast, ATM's repair function is important for survival post irradiation.     

DNA损伤检验点调控的分子机制

多种因素可以引起DNA损伤而最终导致基因产生错义突变、缺失或错误重组。为确保遗传准确性,细胞形成了复杂的细胞周期监督机制,即细胞周期检验点。其中DNA损伤检验点由许多检验点相关蛋白组成,可以识别损伤的DNA,经复杂的信号转导途径引发蛋白激酶的级联反应,减慢或阻滞细胞周期进程,从而为细胞修复损伤的DNA赢得时间。     

A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.     

细胞DNA损伤检控系统在维持端粒稳定中的功能

真核生物的DNA损伤检控系统是维持细胞基因组稳定的一个重要机制,该系统能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,对DNA损伤进行修复,以维持细胞遗传的稳定性。端粒是位于真核细胞染色体末端由重复DNA序列和蛋白质组成的复合物,具有保护染色体、介导染色体复制、引导减数分裂时的同源染色体配对和调节细胞衰老等作用。虽然端粒与DNA双链断裂都具有作为线性染色体末端的共同特点,但正常端粒并不像DNA双链断裂那样激活DNA损伤检控系统。另一方面,端粒又与DNA损伤相似,因为多种DNA损伤检控蛋白在端粒长度稳定中起重要作用。因此DNA损伤检控系统既参与了维持正常端粒的完整性,又可对端粒损伤作出应答。现就DNA损伤检控系统在维持端粒稳定中的作用及其对功能缺陷端粒的应答作一简要综述。     

Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.     

Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast

Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents. We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest. Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entrée into some forms of genomic instability. Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants. This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest. Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.     

DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.     

The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint

Eukaryotic genome integrity is maintained via a DNA damage checkpoint that recognizes DNA damage and halts the cell cycle at metaphase, allowing time for repair. Checkpoint signaling is eventually terminated so that the cell cycle can resume. How cells restart cell division following checkpoint termination is poorly understood. Here we show that the SUMO protease Ulp2 is required for resumption of cell division following DNA damage-induced arrest in Saccharomyces cerevisiae, although it is not required for DNA double-strand break repair. The Rad53 branch of the checkpoint pathway generates a signal countered by Ulp2 activity following DNA damage. Interestingly, unlike previously characterized adaptation mutants, ulp2Delta mutants do not show persistent Rad53 phosphorylation following DNA damage, suggesting checkpoint signaling has been terminated and no longer asserts an arrest in these cells. Using Cdc14 localization as a cell cycle indicator, we show that nearly half of cells lacking Ulp2 can escape a checkpoint-induced metaphase arrest despite their inability to divide again. Moreover, half of permanently arrested ulp2Delta cells show evidence of an aberrant mitotic spindle, suggesting that Ulp2 is required for proper spindle dynamics during cell cycle resumption following a DNA damage-induced cell cycle arrest.     

Molecular anatomy of the DNA damage and replication checkpoints

Cell cycle checkpoints are signal transduction pathways that enforce the orderly execution of the cell division cycle and arrest the cell cycle upon the occurrence of undesirable events, such as DNA damage, replication stress, and spindle disruption. The primary function of the cell cycle checkpoint is to ensure that the integrity of chromosomal DNA is maintained. DNA lesions and disrupted replication forks are thought to be recognized by the DNA damage checkpoint and replication checkpoint, respectively. Both checkpoints initiate protein kinase-based signal transduction cascade to activate downstream effectors that elicit cell cycle arrest, DNA repair, or apoptosis that is often dependent on dose and cell type. These actions prevent the conversion of aberrant DNA structures into inheritable mutations and minimize the survival of cells with unrepairable damage. Genetic components of the damage and replication checkpoints have been identified in yeast and humans, and a working model is beginning to emerge. We summarize recent advances in the DNA damage and replication checkpoints and discuss the essential functions of the proteins involved in the checkpoint responses.     

Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression.

The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

细胞周期检控点

细胞周期是高度有组织的时序调控过程,受到DNA损伤检控点、DNA复制检控点和纺锤体检控点等细胞周期检控点的精确调控。细胞周期检控点的作用主要是调节细胞周期的时序转换,以确保DNA复制、染色体分离等细胞重要生命活动的高度精确性,并对DNA损伤、DNA复制受阻、纺锤体组装和染色体分离异常等细胞损伤及时做出反应,以防止突变和遗传不稳定的发生。细胞周期检控点的功能缺陷,将导致细胞基因组的不稳定,与细胞癌变密切相关。因此细胞周期检控点对于维持细胞遗传信息的稳定性和完整性以及防止细胞癌变和遗传疾病的发生起着至关重要的作用。     

Elevated cyclin G2 expression intersects with DNA damage checkpoint signaling and is required for a potent G2/M checkpoint arrest response to doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.     

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.     

DNA damage-dependent cyclin D1 proteolysis: GSK3β holds the smoking gun

Ubiquitin mediated degradation of cyclin D1 following the G1/S transition counters its mitogen-dependent accumulation during G1 phase of the cell cycle. Although the cellular machinery responsible for this process has been identified, how this regulatory pathway interfaces to cellular stress responses, often referred to as checkpoints, remains to be established. One intensely investigated checkpoint is the cellular response to DNA damage. When DNA damage is sensed, the corresponding DNA damage checkpoint triggers the inhibition of CDK-dependent cell cycle progression, with arrest coordinated by induction of CDK inhibitors and rapid degradation of specific cyclins, such as cyclin D1. In recent work, we identified a phosphorylation- and Fbx4-dependent cyclin D1 degradation mechanism in response to genotoxic stress.18 This work revealed that loss of cyclin D1 regulation compromises the intra-S-phase response to DNA damage, promoting genomic instability and sensitization of cells to S-phase chemotherapy, highlighting a potential therapeutic strategy for cancers exhibiting cyclin D1 accumulation.     

Non-Catalytic Function for ATR in the Checkpoint Response

The ATR family of checkpoint kinases is essential for an appropriate response to genomic insults in eukaryotes. Included in this family are Mei-41 in Drosophila, Mec1 in S. cerevisiae, Rad3 in S. pombe, and ATR in vertebrates. These large kinases phosphorylate and modify multiple cell cycle and checkpoint factors, leading to cell cycle arrest, DNA repair, and induction of apoptosis. The catalytic domain of all ATR family members comprises only a fraction of the total protein. Here, we show that the non-catalytic portion of ATR has a conserved function in the checkpoint response. Expression of either wild type or various kinase defective forms of Xenopus ATR (XATR) in S. cerevisiae mec1 mutants suppresses the checkpoint defect and induces a DNA damage dependent mitotic cell cycle arrest. This suppression requires the presence of yeast Ddc2 and Rad9 but functions independently of Rad9 modification and Rad53 activation. Our results indicate that XATR is not functioning through the established mitotic checkpoint pathways. Instead, we find that the XATR suppression of the mec1 mutant checkpoint defect requires the spindle checkpoint factors Mad1 and Mad2, suggesting a role for XATR in the spindle assembly checkpoint. Finally, we show that a yeast strain expressing a truncated, kinase domain deleted form of mec1 from the endogenous locus is partially checkpoint proficient and induces a mitotic cell cycle arrest in a Mad2 dependent manner. Thus, the link between the non-catalytic region of the ATR kinase family and the spindle checkpoint pathway is conserved.     

The E3 Ubiquitin Ligase EDD Regulates S-Phase and G2/M DNA Damage Checkpoints

The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G1/S and intra S phase DNA damage checkpoint activation and for the maintenance of G2/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability, emphasising the potential importance of dysregulated EDD expression and/or function in the evolution of cancer.     

Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation.

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.     

Dysfunctional Telomeres at Senescence Signal Cell Cycle Arrest via Chk2

Loss of telomere integrity can have two outcomes with opposite predicted effects on tumorigenesis. On the one hand, shortened telomeres in normal cells may trigger cell cycle arrest, leading to tumour suppression. On the other hand, in a tumour cell in which neither the p53 nor pRb pathway is intact, shortened telomeres could initiate chromosome instability and promote tumorigenesis A major issue in telomere research is to understand how shortened dysfunctional telomeres can regulate the onset of cellular senescence. Recent studies have revealed that critically shortened or acutely uncapped telomeres share molecular features with damaged DNA. We have recently linked the phosphorylation and activation of one major DNA damage effector checkpoint kinase, Chk2, to telomere erosion in signalling cell cycle arrest in normal fibroblasts. Here, we discuss several hypotheses to explain the molecular events occurring at shortened telomeres that ultimately lead to cell cycle arrest or increased genomic instability.