G-四联体的生物学功能研究进展

G-四联体是一种特殊的核酸二级结构,广泛存在于人类基因组DNA以及RNA中,如DNA的端粒序列、基因的启动子序列、RNA的5'端非翻译区(5'UTR)序列等。许多研究发现,G-四联体结构在基因的稳定性、端粒合成过程、基因转录和翻译水平的表达调控、基因重组等生命过程中起着至关重要的作用。目前的研究主要涉及DNA中的G-四联体、RNA中的G-四联体以及人工设计的G-四联体寡聚核苷酸,本文将从这三个方面介绍G-四联体的生物学功能相关研究进展。     

G-四链体核酸的生物学功能

G-四链体是由富含鸟嘌呤的DNA或RNA折叠形成的高级结构。可形成G-四链体的序列在人基因组中广泛分布,涉及DNA复制、端粒维持、基因表达与调控以及遗传不稳定性等过程。研究发现有些化学合成的G-四链体序列也具有生物活性,如核仁素的核酸适体AS1411具有抑制恶性肿瘤增殖活性。G-四链体的生物学功能研究对于恶性疾病的发病机理和靶向治疗,以及设计开发核酸类抗癌药物有重要意义。     

G-四链体结构的检测、功能及调控

G-四链体结构是近年来发现的特殊核酸二级结构,它在体内极易形成,分布十分广泛并且具有重要的生物学功能。研究者们已经在体外检测到G-四链体的存在并解析出其晶体结构,各种检测该结构的方法如特异性荧光探针、抗体等也不断被发现或合成。G-四链体不仅广泛分布于端粒、启动子区、外显子等具有重要功能的基因区域,在5'非编码区(5'UTR)、内含子区、3'非编码区(3'UTR)等也有广泛存在。相应区域的G-四链体参与到端粒延长、DNA复制、转录、减数分裂、基因重组等重要的生命过程,发挥抗肿瘤、抗病毒、抑制血管新生等作用。目前基于G-四链体结构的抗肿瘤药物已经进入临床试验阶段并取得了良好的疗效。G-四链体结构的内源性调节包括多种内源性蛋白以及碱基的甲基化等,维持其含量与结构的平衡状态。此外,外源性小分子也可对体内G-四链体的平衡状态发挥调节作用。本文将从化学、生物和医学的角度对G-四链体结构的检测方法及其特殊功能和调控进行系统的论述和展望。     

G- 四链体DNA 结合剂研究进展

富含鸟嘌呤的单链DNA序列可以缠绕折叠形成G- 四链体结构。人类基因组中有36,000 个以上的DNA 序列有潜力生成 G-四链体,如端粒末端重复序列,以及c-myc、c-kit、bcl-2 等原癌基因启动子区域。G-四链体是由四个鸟嘌呤之间通过Hoogsteen 氢键形成G-四分体,相邻的G-四分体再通过π-π 堆积作用,由糖- 磷酸骨架相连而成。G- 四链体DNA 的形成有着重要的生 物学意义,它和相关基因表达水平密切相关,诱导和稳定G- 四链体结构就有可能抑制癌基因的转录和表达,引起肿瘤细胞生物 学功能的紊乱,从而抑制肿瘤细胞的增殖。G-四链体结构作为新的抗肿瘤药物靶点引起了科学家的广泛关注,能够稳定G- 四链 体结构的配体包括二酰胺蒽醌类、苝类、阳离子卟啉类、金属配合物和天然产物等。本文对近年来以G-四链体为靶点的配体的研 究进行了综述。     

G-四链体DNA结合剂研究进展

富含鸟嘌呤的单链DNA序列可以缠绕折叠形成G-四链体结构。人类基因组中有36,000个以上的DNA序列有潜力生成G-四链体,如端粒末端重复序列,以及c-myc、c-kit、bcl-2等原癌基因启动子区域。G-四链体是由四个鸟嘌呤之间通过Hoogsteen氢键形成G-四分体,相邻的G-四分体再通过π-π堆积作用,由糖-磷酸骨架相连而成。G-四链体DNA的形成有着重要的生物学意义,它和相关基因表达水平密切相关,诱导和稳定G-四链体结构就有可能抑制癌基因的转录和表达,引起肿瘤细胞生物学功能的紊乱,从而抑制肿瘤细胞的增殖。G-四链体结构作为新的抗肿瘤药物靶点引起了科学家的广泛关注,能够稳定G-四链体结构的配体包括二酰胺蒽醌类、苝类、阳离子卟啉类、金属配合物和天然产物等。本文对近年来以G-四链体为靶点的配体的研究进行了综述。     

癌症治疗的目标——抑制端粒酶的策略

端粒酶与癌症密切相关,抑制端粒酶的活性可以抑制癌细胞的生长.反义核酸、核酶、细胞分化剂、逆转录酶抑制剂和鸟嘌呤四联体等都可以在不同程度上抑制端粒酶活性.在癌症治疗中具有很大应用潜力.     

癌症治疗的目的

端粒酶与癌症密切相关,抑制端粒酶的活性主抑制癌细胞的生长,反义核酸、核酶、细胞分化剂、逆录酶抑制剂和鸟嘌呤四联体等都可以在不同程度上抑制端粒酶活性,在癌症治疗中具有很大应用潜力。     

G-四链体与配体相互作用的研究方法进展

核酸的G-四链体结构在原核生物和真核生物的基因组中广泛存在,并参与基因复制和重组、端粒延伸、基因表达调控等多种重要的生物学过程.G-四链体与配体如Telomestatin、TMPy P4、BRACO-19、RHPS4等的相互作用研究有助于阐明其生物学功能.G-四链体与配体分子间的相互作用研究应用多种分析方法,如硫酸二甲酯印迹、凝胶迁移、聚合酶终止实验等生物化学法,而现代分析技术包含圆二色谱、荧光光谱、荧光共振能量转移、核磁共振、X-射线晶体衍射等光谱法,以及表面等离子体共振、电喷雾质谱和毛细管电泳法等.本文综述了可与G-四链体结合的配体以及G-四链体与配体相互作用的研究方法,并对各种方法进行了比较.     

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。     

DNA的G—四联体螺旋结构

ＤＮＡ的Ｇ┐四联体螺旋结构刘定燮王昌才（第一军医大学分子生物学研究所,广州５１０５１５）关键词ＤＮＡＧ－四联体螺旋端粒是由独特的ＤＮＡ序列及相关蛋白质组成的线性真核染色体末端结构,它参与稳定染色体末端及其精确复制等过程。端粒ＤＮＡ一般由富含Ｇ的简单重...     

Telomere fusion to chromosome breaks reduces oncogenic translocations and tumour formation

Telomeres protect chromosome ends from fusion, degradation and recombination. Loss of telomere function has opposite effects on tumorigenesis: apoptosis, which inhibits tumour growth, and genomic instability, which accelerates tumour formation. Here we describe a new mechanism by which short telomeres inhibit tumorigenesis through interference with oncogenic translocations. In mice that are null for both ataxia-telangiectasia-mutated (Atm) and telomerase RNA (mTR), the first generation (G1) Atm-/- mTR-/- mice have a lower rate of tumour formation than Atm-/- mTR+/+ mice. These Atm-/- mTR-/- G1 tumours show no increase in either apoptosis or overall genomic instability. Strikingly, the tumours show a high fraction of translocations containing telomere signals at the translocation junctions. Translocations of the T-cell receptors on chromosome 14, which initiate tumorigenesis, were interrupted by fusion with telomeres. Telomere repeats were also detected at the translocation junctions in pre-malignant thymocytes. We propose that telomere fusion to DNA double-strand breaks competes with the generation of oncogenic translocations and thus reduces tumour formation.     

An exonuclease I hydrolysis assay for evaluating G-quadruplex stabilization by small molecules

Telomere length homeostasis is a prerequisite for the generation and growth of cancer. In >85% cancer cells, telomere length is maintained by telomerase that add telomere repeats to the end of telomere DNA. Because the G-rich strand of telomere DNA can fold into G-quadruplex that inhibits telomerase activity, stabilizing telomere quadruplex by small molecules is emerging as a potential therapeutic strategy against cancer. In these applications, the specificity of small molecules toward quadruplex over other forms of DNA is an important property to ensure no processes other than telomere elongation are interrupted. The evaluating assays currently available more or less have difficulty identifying or distinguishing quadruplex-irrelevant effect from quadruplex stabilization. Here, we describe an exonuclease I hydrolysis assay that evaluates quadruplex stabilization by DNA-interacting compounds, discriminates inhibitory effect from different sources and helps determine the optimal compound concentration.     

Structure of hnRNP D complexed with single-stranded telomere DNA and unfolding of the quadruplex by heterogeneous nuclear ribonucleoprotein D

Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG)n, the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3'-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase.     

DNA adducts of antitumor cisplatin preclude telomeric sequences from forming G quadruplexes

We studied the effect of antitumor cisplatin and inefficient transplatin on the structure and stability of G quadruplexes formed by the model human telomere sequence 5′-GGG(TTAGGG)₃-3′ using circular dichroism, UV-monitored thermal denaturation, and gel electrophoresis. In addition, to investigate whether there is a connection between the ability of cisplatin or transplatin to affect telomerase activity and stability of G quadruplexes, we also used a modified telomere repeat amplification protocol assay that uses an oligonucleotide substrate for telomerase elongation susceptible to forming a G quadruplex. The results indicate that cisplatin is more efficient than transplatin in disturbing the quadruplex structure, thereby precluding telomeric sequences from forming quadruplexes. On the other hand, the results of this work also demonstrate that in absence of free platinum complex, DNA adducts of antitumor cisplatin inhibit telomerase catalysis, so the mechanism underlying this inhibition does not involve formation of the G quadruplexes which are not elongated by telomerase.     

Targeting telomerase and telomeres: a click chemistry approach towards highly selective G-quadruplex ligands

Maintenance of telomeres--specialized complexes that protect the ends of chromosomes, is undertaken by the enzyme complex telomerase, which is a key factor that is activated in more than 80% of cancer cells, but is absent in most normal cells. Targeting telomere maintenance mechanisms could potentially halt tumour growth across a broad spectrum of cancer types, with little cytotoxic effect outside cancer cells. Here, we describe in detail a new class of G-quadruplex binding ligands synthesized using a click chemistry approach. These ligands comprise a 1,3-di(1,2,3-triazol-4-yl)benzene pharmacophore, and display high levels of selectivity for interaction with G-quadruplex DNA vs. duplex DNA. The ability of these ligands to inhibit the enzymatic activity of telomerase correlates with their ability to stabilize quadruplex DNA, and with estimates of affinity calculated by molecular modeling.     

Est1 protects telomeres and inhibits subtelomeric y'-element recombination

In the budding yeast Saccharomyces cerevisiae, the structure and function of telomeres are maintained by binding proteins, such as Cdc13-Stn1-Ten1 (CST), Yku, and the telomerase complex. Like CST and Yku, telomerase also plays a role in telomere protection or capping. Unlike CST and Yku, however, the underlying molecular mechanism of telomerase-mediated telomere protection remains unclear. In this study, we employed both the CDC13-EST1 fusion gene and the separation-of-function allele est1-D514A to elucidate that Est1 provided a telomere protection pathway that was independent of both the CST and Yku pathways. Est1's ability to convert single-stranded telomeric DNA into a G quadruplex was required for telomerase-mediated telomere protection function. Additionally, Est1 maintained the integrity of telomeres by suppressing the recombination of subtelomeric Y' elements. Our results demonstrate that one major functional role that Est1 brings to the telomerase complex is the capping or protection of telomeres.     

Modelling the regulation of telomere length: the effects of telomerase and G-quadruplex stabilising drugs

Telomeres are guanine-rich sequences at the end of chromosomes which shorten during each replication event and trigger cell cycle arrest and/or controlled death (apoptosis) when reaching a threshold length. The enzyme telomerase replenishes the ends of telomeres and thus prolongs the life span of cells, but also causes cellular immortalisation in human cancer. G-quadruplex (G4) stabilising drugs are a potential anticancer treatment which work by changing the molecular structure of telomeres to inhibit the activity of telomerase. We investigate the dynamics of telomere length in different conformational states, namely t-loops, G-quadruplex structures and those being elongated by telomerase. By formulating deterministic differential equation models we study the effects of various levels of both telomerase and concentrations of a G4-stabilising drug on the distribution of telomere lengths, and analyse how these effects evolve over large numbers of cell generations. As well as calculating numerical solutions, we use quasicontinuum methods to approximate the behaviour of the system over time, and predict the shape of the telomere length distribution. We find those telomerase and G4-concentrations where telomere length maintenance is successfully regulated. Excessively high levels of telomerase lead to continuous telomere lengthening, whereas large concentrations of the drug lead to progressive telomere erosion. Furthermore, our models predict a positively skewed distribution of telomere lengths, that is, telomeres accumulate over lengths shorter than the mean telomere length at equilibrium. Our model results for telomere length distributions of telomerase-positive cells in drug-free assays are in good agreement with the limited amount of experimental data available.     

Fluorescence-based melting assays for studying quadruplex ligands

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for T(m) calculation, complicating a direct comparison of the results obtained by different laboratories.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

端粒酶活性调节的分子机制

人端粒酶由RNA亚基、hTERT催化亚基和hTEP1调节蛋白等组成。端粒酶对端粒结构的稳定起着重要的作用,而端粒结构和端粒结合蛋白也影响着端粒酶活性。某些化疗药物通过破坏端粒结构下调端粒酶活性。端粒酶的激活需要hTERT基因的从头转录和各个蛋白亚基正确装配为端粒酶全酶。端粒酶活性调节的分子机制包括：（1）TERT基因的表达和转录是决定端粒酶活性的重要环节,受多种因素调控;（2）蛋白激酶Cα和蛋白激酶B磷酸化端粒酶蛋白而激活端粒酶,蛋白磷酸酯酶2A（PP2A）可逆转这一过程,下调端粒酶活性;（3）多种癌基因和抑癌基因及其编码的蛋白质也直接或间接与端粒蛋白、端粒酶蛋白反应,参与端粒酶活性的调控。