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。     

Telomere uncapping during in vitro T-lymphocyte senescence

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in γ-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16 ^ink4a upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1 , hTANK1 , hTIN2 , hPOT1 and hRAP1 , was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.     

端粒和端粒酶的发现及其生物学意义

2009年的诺贝尔生理学或医学奖授予了美国加州大学旧金山分校的Elizabeth H．Blackburn、约翰霍普金斯大学的Carol W．Greider以及哈佛医学院的Jack W．Szostak三位科学家,肯定他们在发现端粒以及端粒酶保护染色体末端方面所做出的贡献。端粒以及端粒酶的发现历经近半个世纪,追溯起端粒和端粒酶的整个发现过程,却是耐人寻味,给人启发。端粒是真核生物中位于染色体末端的DNA和蛋白质的复合物,它对于维持基因组的完整性以及染色体的稳定性都有着至关重要的作用。端粒DNA可以被一种特化的称为“端粒酶”的逆转录酶延伸。端粒长度的维持以及端粒结构的稳定在细胞衰老、癌症发生以及干细胞全能性自我更新能力维持等生命过程中都起重要作用。     

端粒结合蛋白TRF2的研究进展

端粒DNA结合蛋白TRF2(TTAGGG repeat binding factor-2)以二聚体形式通过Myb结构域与端粒重复序列TTAGGG结合,并与TRF1、TIN2、Rap1、TINT1及POT1蛋白组成Shelterin蛋白复合物,协同在端粒动态平衡维持过程中起关键作用,进而影响整个基因组的稳定性。此外,TRF2在细胞DNA损伤应答过程中可能发挥重要作用。本文将对TRF2结构和功能研究的最新进展进行综述。     

Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects (‘caps'') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1–Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tel1 kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.     

银染端粒重复序列检测端粒酶活性方法的研究

为了建立一种便于检测端粒酶活性的方法,在Kim等开发的TRAP法的基础上作了一些改进。即把细胞提取液与TS引物混合,30度保温30min以延伸TS引物之后经过酚／氯仿抽提,乙醇沉淀,再做PCR扩增,PCR产物经12％非变性聚丙烯酰胺凝胶电泳,用银染法来显示电泳结果,结果表明该方法可以有效去除抑制Taq酶活性的因素,得到清晰的6bp阳性条带,具有特异性好,灵敏度高,易操作,无放射性危害等优点。     

端粒及端粒酶活性的调控机制

广义的端粒由帽子、双链的串联重复序列的DNA核心部分及亚端粒构成,其结合蛋白是一个复合体,由TRF1、TRF2、TIN2、Pot1、TPP1、RAP1 6个亚单位组成;另外,还结合组蛋白的特定成分H3K9三甲基聚合体和H4K20三甲基聚合体。端粒酶主要由hTerc、hTert、dyskerin构成。端粒的功能主要受端粒酶的活性调控;而端粒酶活性主要受hTert及hTerc的转录水平和转录后的剪切、hTert的翻译等因素的调控。端粒与端粒酶结构和功能的异常与细胞衰老及肿瘤的发生、发展关系密切。     

A hypomorphic allele of telomerase uncovers the minimal functional length of telomeres in Arabidopsis

Despite the essential requirement of telomeric DNA for genome stability, the length of telomere tracts between species substantially differs, raising the question of the minimal length of telomeric DNA necessary for proper function. Here, we address this question using a hypomorphic allele of the telomerase catalytic subunit, TERT. We show that although this construct partially restored telomerase activity to a tert mutant, telomeres continued to shorten over several generations, ultimately stabilizing at a bimodal size distribution. Telomeres on two chromosome arms were maintained at a length of 1 kb, while the remaining telomeres were maintained at 400 bp. The longest telomeres identified in this background were also significantly longer in wild-type populations, suggesting cis-acting elements on these arms either promote telomerase processivity or recruitment. Genetically disrupting telomerase processivity in this background resulted in immediate lethality. Thus, telomeres of 400 bp are both necessary and sufficient for Arabidopsis viability. As this length is the estimated minimal length for t-loop formation, our data suggest that telomeres long enough to form a t-loop constitute the minimal functional length.     

Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress

Telomerase inhibition may be a novel anti-cancer strategy that can be used in combination with conventional therapies, such as DNA damaging agents. There are conflicting reports as to whether and to what extent telomerase and telomere length influence the sensitivity of cells to genotoxins. To understand the relationship between telomere length, telomerase expression, and sensitivity to genotoxic stress, we expressed the catalytic subunit of telomerase, hTERT, in human fibroblasts having different telomere lengths. We show that telomerase confers resistance to ionizing radiation, bleomycin, hydrogen peroxide, and etoposide only in cells with short, presumably near-dysfunctional, telomeres. This resistance depended on the ability of telomerase to elongate the short telomeres, and telomerase did not protect cells with long telomeres. Interestingly, although long telomeres had no effect on sensitivity to etoposide and bleomycin, they exacerbated sensitivity to hydrogen peroxide, supporting the idea that, compared to other types of DNA damage, telomeres are particularly vulnerable to oxidative damage. Our findings identify a mechanism and conditions under which telomerase and telomeres affect the response of human cells to genotoxic agents and may have important implications for anti-cancer interventions.     

Telomere length analysis in residents of a community exposed to arsenic

Differentiated cells telomere length is an indicator of senescence or lifespan; however, in peripheral blood leukocytes the relative shortening of the telomere has been considered as a biological marker of aging, and lengthening telomere as an associated risk to cancer. Individual’s age, type of tissue, lifestyle, and environmental factors make telomere length variable. The presence of environmental carcinogens such as arsenic (As) influence as causal agents of these alterations, the main modes of action for As described are oxidative stress, reduction in DNA repair capacity, overexpression of genes, alteration of telomerase activity, and damage to telomeres. The telomeres of leukocytes resulting a finite capacity of replication due to the low or no activity of the telomerase enzyme, therefore, elongation telomere in this kind of cells is a potential biological marker associated with the development of chronic diseases and carcinogenesis.     

亚硒酸钠对肝细胞L-02端粒酶活性和端粒长度的作用

通过研究硒对端粒酶活性和端粒长度的作用 ,探讨硒抗衰老的生物学机制。实验以人肝细胞株L 0 2为研究对象 ,分别补充 0 .5和 2 .5 μmol L亚硒酸钠 ,采用端粒重复序列扩增 焦磷酸根酶联发光法、逆转录聚合酶链式反应法及流式荧光原位杂交法 ,分别检测细胞的端粒酶活性、人端粒酶逆转录酶催化亚基基因 (hTERT)的表达及端粒长度的变化。结果表明 :常规培养的肝细胞株L 0 2的端粒酶活性和hTERT基因表达水平均较低。补充 0 .5和2 .5 μmol L亚硒酸钠三周后细胞生长状况良好、端粒酶活性和hTERT基因表达水平显著性增高 ,且呈一定的剂量 效应关系。细胞补充亚硒酸钠四周后端粒长度显著增长。说明营养浓度的亚硒酸钠可通过提高端粒酶活性和增长端粒长度来减缓L 0 2肝细胞衰老、延长细胞寿命。     

Taming the tiger by the tail: modulation of DNA damage responses by telomeres

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell‐cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell‐cycle division are discussed.     

端粒结合蛋白在端粒复制与损伤修复中的作用

端粒位于真核细胞线性染色体末端,正常的端粒长度与结构对于细胞基因组稳定的维持有重要作用.端粒DNA序列的高度重复性使其容易形成一些特殊的二级结构,相比染色体其他位置更难复制.结合在端粒上的Shelterin蛋白复合体由六个端粒结合蛋白组成,该复合体可以通过抑制端粒处异常DNA损伤修复途径的激活维持端粒的稳定.此外,近几...     

铅和硒对端粒长度、端粒酶及端粒结合蛋白的影响

以酿酒酵母细胞为实验材料 ,在分子水平上研究铅 (Pb)和硒 (Se)对端粒长度、端粒酶及端粒结合蛋白的影响。结果发现 :与对照组相比 ,添加 1mg/LPb的培养基中培养 10 0代后的酿酒酵母细胞中端粒DNA的平均长度缩短 ,端粒结合蛋白Rap1p含量减少 ,而且Rap1p蛋白的二级结构受到扰动、端粒酶活性降低、43%的细胞死亡。加 1mg/LSe培养 10 0代后的酿酒酵母细胞与对照组相比 ,细胞中端粒平均长度增加 ,Rap1p蛋白浓度和二级结构保持稳定 ,端粒酶活性增加 ,细胞正常存活。以上结果表明 ,Pb对酿酒酵母细胞端粒有损伤 ,而且其损伤在子代细胞中有累积效应 ;而Se对Pb损伤具有一定程度的修复保护作用 ,适量给机体补Se对抑制细胞损伤和衰老有一定作用。由于端粒的特殊结构特征 ,推断Pb和Se是通过作用于端粒酶及端粒结合蛋白而间接影响端粒长度的     

Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with telomerase

Although telomere‐binding proteins constitute an essential part of telomeres, in vivo data indicating the existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization of a number of candidate proteins has not identified true components of plant shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with POT1b (Protection of telomeres 1). Here we demonstrate co‐localization of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric interactome of A. thaliana.     

Ontogeny of telomerase in chicken: impact of downregulation on pre- and postnatal telomere length in vivo

Telomeres are the termini of linear chromosomes composed of tandem repeats of a conserved DNA sequence. Telomerase provides a mechanism for proliferating cells to offset telomeric sequence erosion by synthesizing new repeats onto the end of each parental DNA strand. Reduced or absent telomerase activity can lead to telomere shortening and genome instability. Telomeres and telomerase have not previously been characterized during ontogeny of any avian species. In the present study, telomerase activity in the chicken model was examined from early differentiation embryos through to adulthood. Telomerase activity was detected in all early embryos (preblastula through neurula) and in tissues throughout organogenesis. Subsequently, telomerase was downregulated in the majority of somatic tissues, either pre- or postnatally. A subset of tissues, such as intestine, immune and reproductive organs, exhibited constitutive activity. The impact of telomerase downregulation on telomere length was investigated and a telomere reduction of 3.2 kb in somatic tissues compared with germ line was observed in 5-year-old adults. The present results suggest that the telomere clock function is a conserved feature of avians as well as mammals. Knowledge regarding the relationships among telomerase regulation, proliferation/senescence profiles and differentiation status will be useful for numerous applications of chicken cells.     

The analysis of telomere length and telomerase activity in cloned pigs and cows

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts.