Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

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。     

Regulation of Claspin degradation by the ubiquitin-proteosome pathway during the cell cycle and in response to ATR-dependent checkpoint activation

Claspin is involved in ATR-dependent activation of Chk1 during DNA replication and in response to DNA damage. We show that degradation of Claspin by the ubiquitin-proteosome pathway is regulated during the cell cycle. Claspin is stabilized in S-phase but is abruptly degraded in mitosis and is absent from early G(1) cells in which the phosphorylation of Chk1 by ATR is abrogated. In response to hydroxyurea, UV or aphidicolin, Claspin is phosphorylated in the Chk1-binding domain and its protein levels are increased in an ATR-dependent manner. Thus, the Chk1 pathway is regulated through both phosphorylation of Claspin and its controlled degradation.     

Genetic analysis of CHK1 and CHK2 homologues revealed a unique cross talk between ATM and ATR pathways in Neurospora crassa

DNA damage checkpoint is an important mechanism for organisms to maintain genome integrity. In Neurospora crassa, mus-9 and mus-21 are homologues of ATR and ATM, respectively, which are pivotal factors of DNA damage checkpoint in mammals. A N. crassa clock gene prd-4 has been identified as a CHK2 homologue, but its role in DNA damage response had not been elucidated. In this study, we identified another CHK2 homologue and one CHK1 homologue from the N. crassa genome database. As disruption of these genes affected mutagen tolerance, we named them mus-59 and mus-58, respectively. The mus-58 mutant was sensitive to hydroxyurea (HU), but the mus-59 and prd-4 mutants showed the same HU sensitivity as that of the wild-type strain. This indicates the possibility that MUS-58 is involved in replication checkpoint and stabilization of stalled forks like mammalian CHK1. Phosphorylation of MUS-58 and MUS-59 was observed in the wild-type strain in response to mutagen treatments. Genetic relationships between those three genes and mus-9 or mus-21 indicated that the mus-9 mutation was epistatic to mus-58, and mus-21 was epistatic to prd-4. These relationships correspond to two signal pathways, ATR-CHK1 and ATM-CHK2 that have been established in mammalian cells. However, both the mus-9 mus-59 and mus-21 mus-58 double mutants showed an intermediate level between the two parental strains for CPT sensitivity. Furthermore, these double mutants showed severe growth defects. Our findings suggest that the DNA damage checkpoint of N. crassa is controlled by unique mechanisms.     

ATR maintains select progenitors during nervous system development

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis.     

Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis

The POT1 (protection of telomeres) protein binds the single-stranded G-rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53-dependent growth arrest and an ATM-dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR-dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATM-dependent DDR. Our results uncover a novel ATR-dependent DDR at telomeres that is normally shielded by POT1 binding to the single-stranded G-overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo.     

Inhibition of Atm and/or Atr disrupts gene silencing on the inactive X chromosome

ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inactive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the maintenance of heterochromatin.     

When heat casts a spell on the DNA damage checkpoints

Peregrine Laziosi (1265–1345), an Italian priest, became the patron saint of cancer patients when the tumour in his left leg miraculously disappeared after he developed a fever. Elevated body temperature can cause tumours to regress and sensitizes cancer cells to agents that break DNA. Why hyperthermia blocks the repair of broken chromosomes by changing the way that the DNA damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) are activated is an unanswered question. This review discusses the current knowledge of how heat affects the ATR–Chk1 and ATM–Chk2 kinase networks, and provides a possible explanation of why homeothermal organisms such as humans still possess this ancient heat response.     

泛素E3连接酶接头蛋白SPOP和MDM2在DNA损伤修复中的作用

DNA损伤修复是维持细胞基因组稳定性和完整性的基础,越来越多的研究发现,E3泛素连接酶在DNA损伤修复中起着重要的作用.该文将介绍DNA损伤修复的机制、DNA损伤修复与疾病的关系、及E3泛素连接酶接头蛋白MDM2和SPOP在DNA损伤修复中的作用.重点围绕DNA损伤修复的两条通路:E3泛素连接酶接头蛋白SPOP与ATM...     

Regulation of Elg1 activity by phosphorylation

ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1''s activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of Δrad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage.     

ATM、ATR和DNA损伤介导的细胞周期阻滞

ATM和ATR属于PIKK家族,是DNA损伤检查点的主要成员。它们被不同类型的DNA损伤所激活,通过磷酸化相应的下游蛋白Chk1和Chk2等,调节细胞周期各个检查点,引起细胞周期阻滞,使DNA损伤得以修复。ATM和ATR在维持基因组的稳定性中起到至关重要的作用。本文着重综述有关ATM和ATR在DNA损伤介导的细胞周期阻滞中发挥的作用以及相互关系的最新研究进展。     

Protein phosphatase 5 is necessary for ATR-mediated DNA repair

Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.     

A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs

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Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway

Polyploid cells result in aneuploidy through aberrant chromosome segregation, possibly leading to tumorigenesis. Although polyploid cells are induced through over-replication by a variety of agents, including DNA-damaging drugs, the mechanisms that induce polyploidy have been hitherto unknown. Here, we show that treatment with bleomycin, a glycopeptide anticancer drug, induces over-replication at low cytotoxic doses. During bleomycin-induced over-replication, mitotic entry is inhibited through tyrosine phosphorylation of CDK1 along the ATM/ATR pathway in the early phase of treatment. Bleomycin-induced over-replication is inhibited by the inhibitors of the ATM/ATR pathway through abrogation of bleomycin-induced G2 arrest, and the ATM/ATR inhibitors promote cell death instead of over-replication. Following the phosphorylation of CDK1, the level of cyclin B1 is decreased in the late phase of treatment. Time-lapse imaging of clone cells that express a live cell marker of endogenous cyclin B1 revealed that cyclin B1 is degraded in G2-arrested cells upon bleomycin treatment. Our findings lead to a model of how the ATM/ATR pathway acts as a molecular switch for regulating cell fates, flipping between cell death via progress into mitosis, and over-replication via sustained G2 arrest upon DNA damage, where cyclin B1 degradation is an important factor for inducing over-replication.