从DNA修复机理看细胞癌变的发生机制

DNA损伤是引起基因突变,导致细胞恶性转化的重要原因．DNA损伤的修复过程非常复杂,是与细胞周期调节、DNA复制和DNA转录等生命活动紧密相连的．首先DNA修复需要细胞周期停滞,避免DNA损伤进入子代细胞．其次,参与DNA转录的某些基因产物参与DNA损伤的识别,有利于转录链的优先修复．最后,DNA修复系统NER、MMR参与损伤修复．上述DNA修复过程任何环节的异常,都将造成DNA修复功能减弱,导致某些功能基因突变,从而导致细胞的恶性转化．     

自噬与DNA损伤修复及其对肿瘤的影响

DNA损伤与肿瘤的发生发展密切相关。当DNA损伤发生时,会触发一系列的损伤应答反应以帮助细胞生存,其中即包括对自噬的诱导。ATM、P53和PARP1等多种参与DNA损伤修复的效应因子通过影响AMPK、mTOR以及一些凋亡蛋白等启动自噬。而作为一种降解途径,自噬则可通过调节DNA修复相关蛋白的水平直接影响同源重组修复、非同源末端连接修复和核苷酸切除修复等促进DNA修复,以及通过维持细胞内稳态间接促进DNA修复,从而在正常细胞的恶性转化和肿瘤耐药等发生机制中扮演重要角色。此外,DNA修复失败时,自噬也可作为一种肿瘤细胞的程序性死亡方式。因此研究自噬通过调节DNA损伤修复而对肿瘤的影响对于理解肿瘤发生的机制和提供治疗思路都有重要意义。     

环磷酰胺致DNA损伤及其防治机制的研究进展

CTX是临床上常用的抗肿瘤药物与免疫抑制剂,其在体内的代谢产物具有很强的烷化作用,可引起DNA链内和链间的交叉连接,干扰转录和复制,造成DNA的损伤。CTX导致DNA损伤的机制与环磷酰胺代谢过程中产生的自由基过量以及代谢产物产生的烷化作用有关,目前减轻CTX造成的DNA损伤的措施主要是提高机体的抗氧化能力和促进机体修复损伤的DNA。DNA损伤会对机体产生一系列变化如导致肿瘤、神经退行性病变、衰老和遗传易感综合症等疾病的发生、发展。本文综述了CTX导致DNA损伤的机制及其防治研究进展,以其为CTX的临床安全用药和今后的科学研究提供思路和依据。     

DNA损伤修复基本方式的研究进展

DNA损伤修复基因可修复由不同原因导致的DNA损伤．从而保护遗传信息的完整性。DNA损伤修复有3种基本形式,即碱基切除修复、核苷酸切除修复和错配修复。本文综述了DNA损伤修复3种基本形式的研究进展情况并讨论了DNA链断裂重组和重接合修复及DNA聚合酶绕道修复DNA损伤。     

DNA损伤修复过程表观遗传学改变

多种化学、物理及生物因素可诱发细胞DNA损伤,损伤后DNA损伤位点被相关损伤感受器识别,激活相应的修复通路进行DNA修复。越来越多的证据表明DNA甲基化状态、蛋白翻译后修饰、染色质重塑、miRNA等修饰方式参与了DNA的损伤修复。文章通过不同损伤修复通路中这些修饰的特点,阐述表观遗传学改变在DNA损伤修复发展过程中的作用机制。     

DNA损伤检验点与损伤修复及基因组稳定性

生物有机体基因组DNA经常会受到内源或外源因素的影响而导致结构发生变化,产生损伤;在长期进化过程中,有机体也相应形成了一系列应对与修复损伤DNA,并维持染色体基因组正常结构功能的机制。其中DNA损伤检验点（DNA damage checkpoint）就是在感应DNA损伤的基础上,对损伤感应信号进行转导,或引起细胞周期的暂停,从而使细胞有足够的时间对损伤DNA进行修复,或最终导致细胞发生凋亡。DNA损伤检验点信号转导途径是一个高度保守的信号感应过程,整个途径大致可以分为损伤感应、信号传递及信号效应3个组成部分。其中3-磷脂酰肌醇激酶家族类成员ATM（ataxia-telangiectasia mutated）和ATR（ataxia-telangiectasia and Rad3-related）活性的增加构成整个途径活化的第一步。它们通过激活下游的效应激酶,Chk2／Chk1,通过协同作用许多其他调控细胞周期、DNA复制、DNA损伤修复及细胞凋亡等过程的蛋白质因子来实现细胞对DNA损伤的高度协调反应。近十几年,随着此领域研究的不断深入,人们逐步揭示了DNA损伤检验点途径发生过程中,各种核心组分通过与不同调节因子、效应因子及DNA损伤修复蛋白间的复杂相互作用,以实现监测感应异常DNA结构并实施相应反应的机制;其中,检验点衔接因子（mediators）及染色质结构,尤其是核小体组蛋白的共价修饰在调控ATM／ATR活性,促进ATM／ATR与底物间的相互作用以及介导DNA损伤位点周围染色质区域上多蛋白复合物在时间与空间上的动态形成发挥着重要的作用。同时,人们也开始发现DNA损伤检验点途径与DNA损伤修复、基因组稳定性以及肿瘤发生等过程之间某些内在的联系。该反应途径在通过协调细胞针对DNA损伤做出各种反应的基础上,直接或间接地参与或调控DNA损伤修复过程,并与DNA损伤修复途径协同作用最终保证染色体基凶组结构的完整性,而检验点途径的改变,则会引起基因组不稳定的发生,包括从突变频率的提高到大范围的染色体重排,以及染色体数量的畸变。如：突变发生在肿瘤形成早期,会大大增加肿瘤发生的几率。文章将对DNA损伤检验点途径机制及其对DNA损伤修复、基因组稳定性影响的最新进展进行综述。     

调控DNA损伤修复基因的微小RNA

随着对DNA损伤修复基因研究的深入,其信号转导路径及调控网络也进一步明了,调控DNA损伤修复基因的微小RNA(miRNA)也越来越多地被认识和发现。简要综述了DNA损伤途径中调控主要的损伤修复基因的miRNA,有助于深入阐明DNA损伤修复机制,为开发抗辐射药物和临床上DNA损伤修复异常相关肿瘤的基因治疗提供新的靶点。     

Sirtuin家族与DNA损伤修复

DNA损伤的发生与积累是造成细胞功能紊乱的根本原因,也是引起衰老与肿瘤等疾病发生的关键事件。为维持机体自身遗传物质的完整性与稳定性,生物体内拥有多种针对不同类型DNA损伤的修复方式。Sirtuin蛋白是一组NAD+依赖的、高度保守的组蛋白去乙酰化酶,可通过去乙酰化作用调节众多底物蛋白质的表达、活性与稳定性。 近来的研究显示,DNA损伤修复途径的多个关键蛋白质是Sirtuin的下游底物。Sirtuin蛋白通过调节同源重组修复、非同源末端修复、核苷酸切除修复等途径中的核心蛋白质参与修复包括双链断裂（double stranded breakes, DSBs）在内的多种DNA损伤类型,从而在维持基因组稳定性、寿命以及细胞能量代谢调节等一系列生物学作用中发挥至关重要的作用。本综述将介绍近年来Sirtuin与DNA损伤修复的研究进展。     

Sirtuins家族在DNA损伤修复中的作用

Sirtuins家族蛋白是一类依赖NAD的去乙酰化酶,属于第Ш类去乙酰化酶(HDACs),哺乳动物Sirtuins家族成员共有7个(SIRT1-7),其主要具有去乙酰化酶的活性,可以使多种蛋白发生去乙酰化,进而参与DNA的损伤修复、基因的转录调控、细胞凋亡、代谢及衰老等诸多生物进程。本文主要对Sirtuins家族在DNA损伤修复中的作用及其相关机制进行阐述。     

DNA双链断裂损伤修复系统研究进展

多种内源或外源因素都能造成细胞基因组DNA损伤,细胞内建立了复杂的修复系统来应对不同形式的损伤。其中DNA双链断裂(DNA double-strand breaks,DSBs)作为最严重的损伤形式,主要激活同源重组修复(Homologous recombination repair)和非同源末端连接(Non-homologous end joining)通路。这两条通路都是由多个修复元件参与、经过多步反应的复杂过程。两者各具特点、协同作用,共同维护细胞基因组的稳定性。对其分子机制的阐明为肿瘤放化疗的辅助治疗提供了潜在的作用靶点。     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria

In temperate lakes, asynchronous cycles in surface water temperatures and incident ultraviolet (UV) radiation expose aquatic organisms to damaging UV radiation at different temperatures. The enzyme systems that repair UV‐induced DNA damage are temperature dependent, and thus potentially less effective at repairing DNA damage at lower temperatures. This hypothesis was tested by examining the levels of UV‐induced DNA damage in the freshwater crustacean Daphnia pulicaria in the presence and absence of longer‐wavelength photoreactivating radiation (PRR) that induces photoenzymatic repair (PER) of DNA damage. By exposing both live and dead (freeze‐killed) Daphnia as well as raw DNA to UV‐B in the presence and absence of PRR, we were able to estimate the relative importance and temperature dependence of PER (light repair), nucleotide excision repair (NER, dark repair), and photoprotection (PP). Total DNA damage increased with increasing temperature. However, the even greater increase in DNA repair rates at higher temperatures led net DNA damage (total DNA damage minus repair) to be greater at lower temperatures. Photoprotection accounted for a much greater proportion of the reduction in DNA damage than did repair. Experiments that looked at survival rates following UV exposure demonstrated that PER increased survival rates. The important implication is that aquatic organisms that depend heavily on DNA repair processes may be less able to survive high UV exposure in low temperature environments. Photoprotection may be more effective under the low temperature, high UV conditions such as are found in early spring or at high elevations.     

Crosstalk between autophagy and DNA repair systems

Autophagy and DNA repair are two essential biological mechanisms that maintain cellular homeostasis. Impairment of these mechanisms was associated with several pathologies such as premature aging, neurodegenerative diseases, and cancer. Intrinsic or extrinsic stress stimuli (e.g., reactive oxygen species or ionizing radiation) cause DNA damage. As a biological stress response, autophagy is activated following insults that threaten DNA integrity. Hence, in collaboration with DNA damage repair and response mechanisms, autophagy contributes to the maintenance of genomic stability and integrity. Yet, connections and interactions between these two systems are not fully understood. In this review article, current status of the associations and crosstalk between autophagy and DNA repair systems is documented and discussed.     

Space radiation effects and microgravity

Humans in space are exposed both to space radiation and microgravity. The question whether radiation effects are modified by microgravity is an important aspect in risk estimation. No interaction is expected at the molecular level since the influence of gravity is much smaller than that of thermal motion. Influences might be expected, however, at the cellular and organ level. For example, changes in immune competence could modify the development of radiogenic cancers. There are no data so far in this area. The problem of whether intracellular repair of radiation-induced DNA lesions is changed under microgravity conditions was recently addressed in a number of space experiments. The results are reviewed; they show that repair processes are not modified by microgravity.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

Distinct Roles of Ape1 Protein,an Enzyme Involved in DNA Repair,in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.     

Functions and regulation of human artemis in double strand break repair

Cells, which lacked the activity of the nuclease Artemis, retained approximately 10% of unrepaired double strand breaks (DSBs) at later timepoints after ionizing radiation. Ionizing radiation induced hyperphosphorylation of Artemis mainly by ATM and in ATM deficient cells to a minor extent by DNA PK. After induction of DSBs with modified ends by a high dose of calicheamicin gamma1, Artemis was phosphorylated by DNA PK. The type of calicheamicin gamma1-induced DSBs is likely to represent a subclass of DSBs induced by ionizing radiation. DNA PK-dependent phosphorylation of Artemis after treatment with DSB inducing agents increased the cellular retention of Artemis, maintained its interaction with DNA ends and activated its endonucleolytic activity. The following model is suggested: ATM-dependent phosphorylation of Artemis after ionizing radiation could prevent DNA PK-dependent phosphorylation and activation of undesired endonucleolytic activity at DSBs, which do not require endonucleolytic processing by Artemis. The Artemis:DNA PK complex could be involved in the repair of DSBs, which carry modified ends and are refractory to repair by otherwise lesion specific enzymes because of the presence of an inhibitory lesion in the opposite strand.     

DNA修复——基因组稳定性护卫机制的原创与发现之旅

基因组DNA是遗传的物质基础,编码的信息指导生物种系的复制延续、生命体的生长发育和代谢活动。无论是在外环境因素的应激压力下还是处于正常状态,DNA损伤时刻在发生,由此,DNA损伤修复作为重要的细胞内在机制,在维护基因组稳定性、降低癌症等人类系列重大疾病风险中发挥了不可替代作用。三位科学家汤姆·林达尔(Tomas Lindahl)、阿齐兹·桑贾尔（Aziz Sancar）、保罗·莫德里奇（Paul Modrich）因发现和揭示DNA修复及其机制的杰出贡献,获得2015年诺贝尔化学奖。本文综述了三位获奖者分别在DNA损伤的碱基切除修复、核苷酸切除修复和错配修复研究中的原创发现,以及相应的修复通路机制的描绘。此3种修复通路,主要是针对紫外线和化学物所致DNA的碱基损伤、嘧啶二聚体及加合物或者DNA复制过程中发生的碱基错误配对的修复。恰巧,2015年拉斯克基础医学研究奖授予的两位科学家,也因他们揭示了DNA损伤应答现象和机制研究的重大贡献而获奖,本文也呈现了获奖者的关键性科学发现。最后,简要展望了中国DNA损伤修复领域的发展。