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。     

人细胞端粒DNA复制与长度维持

端粒位于染色体末端,由短的串联重复DNA片段及其结合蛋白组成。端粒在维持基因组稳定性及染色体结构完整性方面发挥着重要作用。端粒DNA由富含G/C的序列构成,包括双链区及G含量高的3'悬垂单链区(G-overhang,G-tail)。端粒DNA能够形成G四联体(G-quadruplex)和T环(T-loop)等高级结构。许多与DNA损伤修复相关的蛋白质参与端粒DNA的复制与端粒结构的维持,并相对于基因组的其他区域,端粒的DNA复制较为特别,从广义上讲,端粒DNA的复制可以包括双链复制(telomere replication),端粒酶复制延伸(telomerase extension)和C链补齐(C-rich fill-in)。端粒双链复制引起的端粒长度缩短是导致人体细胞衰老的重要原因,而端粒酶复制延伸及C链补齐是干细胞及肿瘤细胞维持其端粒长度及持续分裂能力的主要途径。端粒复制及其结构功能研究是生物医学领域的一个重要热点,阐释端粒复制的机理将为疾病预防及治疗等提供新的思路。     

端粒DNA损伤与细胞衰老的研究进展

细胞衰老是生物不可逃避的生命现象。研究表明,端粒DNA的长度与细胞的衰老进程有关,衰老细胞的端粒DNA出现不同程度的损伤,如端粒DNA的断裂、融合、缩短和缺失等。因此,端粒长度被称作控制寿命的"生命时钟"。目前,端粒DNA损伤发生的机制也得到进一步阐明。端粒酶作为逆转录酶,主要维持端粒的长度、减少染色体的损伤,保证细胞分裂周期的持续进行。该文探讨了端粒DNA损伤发生的机制及不同类型的端粒DNA损伤与细胞衰老之间的关系,在分子水平上寻找诱发细胞衰老的原因,从而为基础研究转化为临床应用提供思路,为研发相应衰老通路的阻滞剂或端粒酶的激活剂奠定理论基础。     

染色体端粒研究进展

染色体端粒(telomere)是真核生物线性染色体两端的特殊DNA--蛋白质复合体结构,由随机重复序列组成的DNA序列和与之相结合的蛋白质分子构成。端粒DNA无论在DNA顺序、功能及其特殊的复制方式都与其它DNA顺序显著不同。本文将近年来对端粒的DNA结构、与端粒DNA相结合的蛋白质分子和在端粒复制中起重要作用的反转录酶--端粒酶(telomerase)的研究进展以及端粒对于真核生物的重要作用作一综述。     

牛绝对端粒长度测定及DNA提取造成的影响

目的：端粒是真核生物染色体末端的一种高度保守的负责维持染色体稳定的特殊结构,其DNA序列长度即端粒长度,会随着年龄增长或疾病发生发展而逐渐缩短,检测端粒长度可以为评估机体衰老和健康状况提供参考,但目前缺乏测定微量牛DNA样本绝对端粒长度的方法;通过实时荧光定量PCR(real-time quantitative PCR, qPCR)实现微量牛DNA样本绝对端粒长度的测定并评估DNA提取方法对牛绝对端粒长度测定结果的影响,为进行端粒长度研究时选择合适的DNA提取方法和端粒长度分析方法提供参考。方法：利用标准曲线对检测样本的端粒和内参Ct值进行转换,通过qPCR测定牛端粒长度绝对值;采用膜吸附法、苯酚-氯仿法和磁珠法3种方法分别提取相同样本的DNA,分别用端粒末端限制性片段(terminal restriction fragment, TRF)分析法和qPCR法分析端粒长度,比较不同DNA提取方法对牛绝对端粒长度测定的影响。结果：(1)qPCR可以测定纳克级别DNA样本的绝对端粒长度,检测结果重复性良好,并且和“金标准”TRF测定结果的相关性良好。(2)不同方法提取的DNA用TRF分析法和...     

端粒延长替代机制与DNA修复途径

端粒长度和结构的稳定与肿瘤及衰老的发生密切相关,端粒维持机制(telomere maintenance mechanism,TMM)的激活对稳定基因组和建立细胞永生化至关重要。85%~90%的肿瘤细胞是通过激活端粒酶来维持端粒长度的,10%~15%的肿瘤细胞在端粒酶失活或不足的情况下,利用同源重组或其他多种机制维持端粒长度,这些端粒维持机制统称为端粒延长替代机制(alterative lengthening of telomere,ALT)。ALT端粒DNA通过染色体外游离的端粒重复DNA来合成。这提示,在ALT端粒维持时进行的DNA修复机制可能有利于阐明衰老与肿瘤之间的辩证关系。该文从ALT端粒DNA维持的角度,阐述和总结了ALT肿瘤中几种DNA修复途径及ALT活性相关蛋白如何维持端粒长度和功能的完整性。     

靶向端粒抗肿瘤药物的研究进展

端粒是真核生物线性染色体末端的特殊结构,由端粒重复序列DNA和端粒结合蛋白组成,具有保护线性DNA末端、维持染色体稳定性等重要作用。随着细胞分裂次数的增加,端粒长度会逐渐缩短,最终引起细胞衰老或凋亡。端粒长度的维持对持续分裂的肿瘤细胞具有重要的意义。所以,端粒是肿瘤治疗的一个重要靶点,端粒长度维持机制及其相关靶向药物(端粒酶抑制剂、端粒延伸替代机制阻断剂、端粒G-四链体稳定剂以及端粒类似物T-oligo)的研究对于肿瘤治疗有着重要指导意义及临床应用价值。本文聚焦上述四个方向,对以端粒为靶点的抗肿瘤药物的最新研究进展进行综述。     

端粒保护蛋白TRF2的研究进展

端粒是染色体末端的核蛋白结构。染色体末端重复的端粒DNA可以规避不适当的DNA损伤反应(DNA damage response, DDR)的激活,维持染色体的稳定性,端粒的缺失会引起染色体融合并导致细胞的衰老及死亡。端粒特异性蛋白复合物Shelterin在保护端粒完整性方面具有重要作用。在这个复合体中,端粒结合因子2 (telomeric-repeat binding factor 2, TRF2)在维持端粒稳定、防止端粒染色体末端融合以及端粒染色体复制过程中发挥关键作用。该文综述了TRF2介导的保护染色体末端的多方面的机制。     

端粒,端粒酶与癌症关系研究的进展

Muller早于1938年便发现了端粒(Telomere)。1978年Blackburn发现四膜虫染色体端粒为含有(CCCAA,TTGGGG)n重复的一段DNA,但是其功能尚不清楚。1986年端粒酶(Telomerase)的发现,在解决真核生物DNA复制难题的同时,也逐渐揭示了端粒的功能。端粒除保证DNA完整复制外,在维持染色体结构稳定(保护染色体不分解和染色体重排及未端不相互融合等)、染色体在细胞中     

端粒、端粒酶结构功能研究进展

端粒是真核生物线性染色体末端由重复DNA序列和蛋白质结合形成的复合结构,其特殊的环形结构与多种结合蛋白形成了端粒的多重功能的基础。端粒的功能包括染色体末端的保护、引导减数分裂的同源染色体配对、参与DNA修复过程等;端粒酶具有逆转录酶特性和维持端粒长度的功能,其活性与恶性肿瘤的发生密切相关,调控因子错综复杂。     

Arabidopsis ATM and ATR kinases prevent propagation of genome damage caused by telomere dysfunction

The ends of linear eukaryotic chromosomes are hidden in nucleoprotein structures called telomeres, and loss of the telomere structure causes inappropriate repair, leading to severe karyotypic and genomic instability. Although it has been shown that DNA damaging agents activate a DNA damage response (DDR), little is known about the signaling of dysfunctional plant telomeres. We show that absence of telomerase in Arabidopsis thaliana elicits an ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR)-dependent DDR at telomeres, principally through ATM. By contrast, telomere dysfunction induces an ATR-dependent response in telomeric Conserved telomere maintenance component1 (Ctc1)-Suppressor of cdc thirteen (Stn1)-Telomeric pathways in association with Stn1 (CST)-complex mutants. These results uncover a new role for the CST complex in repressing the ATR-dependent DDR pathway in plant cells and show that plant cells use two different DNA damage surveillance pathways to signal telomere dysfunction. The absence of either ATM or ATR in ctc1 and stn1 mutants significantly enhances developmental and genome instability while reducing stem cell death. These data thus give a clear illustration of the action of ATM/ATR-dependent programmed cell death in maintaining genomic integrity through elimination of genetically unstable cells.     

DNA damage foci at dysfunctional telomeres

We report cytologic and genetic data indicating that telomere dysfunction induces a DNA damage response in mammalian cells. Dysfunctional, uncapped telomeres, created through inhibition of TRF2, became associated with DNA damage response factors, such as 53BP1, gamma-H2AX, Rad17, ATM, and Mre11. We refer to the domain of telomere-associated DNA damage factors as a Telomere Dysfunction-Induced Focus (TIF). The accumulation of 53BP1 on uncapped telomeres was reduced in the presence of the PI3 kinase inhibitors caffeine and wortmannin, which affect ATM, ATR, and DNA-PK. By contrast, Mre11 TIFs were resistant to caffeine, consistent with previous findings on the Mre11 response to ionizing radiation. A-T cells had a diminished 53BP1 TIF response, indicating that the ATM kinase is a major transducer of this pathway. However, in the absence of ATM, TRF2 inhibition still induced TIFs and senescence, pointing to a second ATM-independent pathway. We conclude that the cellular response to telomere dysfunction is governed by proteins that also control the DNA damage response. TIFs represent a new tool for evaluating telomere status in normal and malignant cells suspected of harboring dysfunctional telomeres. Furthermore, induction of TIFs through TRF2 inhibition provides an opportunity to study the DNA damage response within the context of well-defined, physically marked lesions.     

The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres

Telomeres protect chromosome ends from being detected as lesions and from triggering DNA damage checkpoints. Paradoxically, telomere function depends on checkpoint proteins such as ATM and ATR, but a molecular model explaining this seemingly contradictory relationship has been missing so far. Here we show that the DNA damage machinery acts on telomeres in at least two independent steps. First, the ATR-dependent machinery is recruited to telomeres before telomere replication is completed, likely in response to single-stranded DNA resulting from replication fork stalling. Second, after replication, telomeres attract ATM and the homologous recombination (HR) machinery. In vivo and in vitro results suggest that the HR machinery is required for formation of a telomere-specific structure at chromosome ends after replication. Our results suggest that telomere ends need to be recognized as DNA damage to complete end replication and to acquire a structure that is essential for function.     

Role of ATM in the telomere response to the G-quadruplex ligand 360A

Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.     

Functional human telomeres are recognized as DNA damage in G2 of the cell cycle

Telomeres have to be distinguished from DNA breaks that initiate a DNA damage response. Proteins involved in the DNA damage response have previously been found at telomeres in transformed cells; however, the importance of these factors for telomere function has not been understood. Here, we show that telomeres of telomerase-negative primary cells recruit Mre11, phosphorylated NBS1, and ATM in every G2 phase of the cell cycle. This recruitment correlates with a partial release of telomeric POT1; moreover, telomeres were found to be accessible to modifying enzymes at this time in the cell cycle, suggesting that they are unprotected. Degradation of the MRN complex, as well as inhibition of ATM, led to telomere dysfunction. Consequentially, we propose that a localized DNA damage response at telomeres after replication is essential for recruiting the processing machinery that promotes formation of a chromosome end protection complex.     

Telomeric protein Pin2/TRF1 as an important ATM target in response to double strand DNA breaks

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiation-induced double strand DNA breaks. Cells derived from A-T patients show many abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA damage. Pin2 was originally identified as a protein involved in G(2)/M regulation and is almost identical to TRF1, a telomeric protein that negatively regulates telomere elongation. Pin2 and TRF1, probably encoded by the same gene, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulation of Pin2 or TRF1 induces mitotic entry and apoptosis, a phenotype similar to that of A-T cells after DNA damage. These results suggest that ATM may regulate the function of Pin2/TRF1, but their exact relationship remains unknown. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its phosphorylation was increased in an ATM-dependent manner by ionizing DNA damage. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser(219)-Gln site in vitro and in vivo. The biological significance of this phosphorylation is substantiated by functional analyses of the phosphorylation site mutants. Although expression of Pin2 and its mutants has no detectable effect on telomere length in transient transfection, a Pin2 mutant refractory to ATM phosphorylation on Ser(219) potently induces mitotic entry and apoptosis and increases radiation hypersensitivity of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser(219) completely fail to induce apoptosis and also reduce radiation hypersensitivity of A-T cells. Interestingly, the phenotype of the phosphorylation-mimicking mutants is the same as that which resulted from inhibition of endogenous Pin2/TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phosphorylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellular response to double strand DNA breaks.     

ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.     

NBS1 deficiency promotes genome instability by affecting DNA damage signaling pathway and impairing telomere integrity

Studies revealed that Nijmegen Breakage Syndrome protein 1 (NBS1) plays an important role in maintaining genome stability, but the underlying mechanism is controversial and elusive. Our results using clinical samples showed that NBS1 was involved in ataxia-telangiectasia mutated (ATM)-dependent pathway. NBS1 deficiency severely affected the phosphorylation of ATM as well as its downstream targets. BrdU proliferation assay revealed a delay of NBS cells in inhibiting DNA synthesis after Doxorubicin (Dox) treatment. In addition, under higher concentrations of Dox, NBS cells exhibited a much lower level of apoptosis compared to their normal counterparts, indicating a resistance to Dox treatment. Accelerated telomere shortening was also observed in NBS fibroblasts, consistent with an early onset of cellular replicative senescence in vitro. This abnormality may be due to the shelterin protein telomeric binding factor 2 (TRF2) which was found to be upregulated in NBS fibroblasts. The dysregulation of telomere shortening rate and of TRF2 expression level leads to telomere fusions and cellular aneuploidy in NBS cells. Collectively, our results suggest a possible mechanism that NBS1 deficiency simultaneously affects ATM-dependent DNA damage signaling and TRF2-regulated telomere maintenance, which synergistically lead to genomic abnormalities.     

MRE11/RAD50/NBS1: complex activities

The MRE11/RAD50/NBS1 complex (Mre11 complex) is a central player in most aspects of the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining, telomere maintenance and DNA damage checkpoint activation. Several of these findings are explained by the unusual enzymatic activities and macromolecular structure of the Mre11 complex. The Mre11 complex possesses an ATP-stimulated nuclease to process heterogeneous DNA ends and long coiled-coil tails to link DNA ends and/or sister chromatids. However, the mechanistic role of the Mre11 complex in checkpoint activation has been unclear until recently. New data suggest that the Mre11 complex can directly activate the ATM checkpoint kinase at DNA breaks. These findings, together with newly determined functional interactions, identify the Mre11 complex as an architectural and mechanistic keystone of cellular response events emerging from DNA breaks.     

ATM is required for telomere maintenance and chromosome stability during Drosophila development

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.