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1、CELL DEATH AND CELL-CYCLE CHECKPOINT DURING DNA DAMAGE细胞死亡及周期阻滞基本信号通路有哪些因素可引起DNA损伤？DNA损伤的结局如何？（课件）(一)DNA损伤的原因环境因素，化学因素，生物因素例如： UV ,离子辐射，基因毒性化学试剂引起ssDNA/dsDNA损伤，DNA两条链交联或链内交联。正常细胞线粒体的一些代谢物（ROS）活泼氧类过多引起损伤。(二) DNA损伤结局：急性效应：干扰核酸代谢，触发细胞周期阻滞或死亡长期效应：不可逆转突变导致肿瘤细胞周期阻滞，衰老，肿瘤/癌症，有丝分裂危象 (一)DNA损伤的原因1.DNA分子的自发性损伤
2、(1)DNA复制中的错误。(2)DNA的自发性化学变化a.碱基的异构互变性损伤b.碱基的脱氨基作用c.脱嘌呤与脱嘧啶d.碱基修饰与链断裂2.物理因素引起的DNA损伤(1)紫外线引起的DNA损伤 (2)电离辐射引起的DNA损伤a.碱基变化b.脱氧核糖变化c.DNA链断裂d.交联3.化学因素引起的DNA损伤(1)烷化剂对DNA的损伤a.碱基烷基化b.碱基脱落c.断链d.交联(2)碱基类似物、修饰剂对DNA的损伤DNA损伤的后果 1.点突变(point mutation)指DNA上单一碱基的变异。嘌呤替代嘌呤(A与G之间的相互替代)、嘧啶替代嘧啶(C与T之间的替代)称为转换(transition)；
3、嘌呤变嘧啶或嘧啶变嘌呤则称为颠换(transvertion)。2.缺失(deletion)指DNA链上一个或一段核苷酸的消失。3.插入(insertion)指一个或一段核苷酸插入到DNA链中。在为蛋白质编码的序列中如缺失及插入的核苷酸数不是3的整倍数，则发生读框移动(reading frame shift)，使其后所译读的氨基酸序列全部混乱，称为移码突变(frameshift mutaion)。4.倒位或转位(transposition)指DNA链重组使其中一段核苷酸链方向倒置、或从一处迁移到另一处。5.双链断裂已如前述，对单倍体细胞一个双链断裂就是致死性事件。（2） THE CONSEQUE
4、NCES OF DNA INJURY The outcome of DNA damage is diverse and generally adverse（有害的）. Acute effects arise from disturbed DNA metabolism（新陈代谢）, triggering（启动，控制） cell-cycle arrest or cell death. Long term effects result from irreversible mutations（转变，突变，变异） contributing to oncogenesis（）. Many Lesions（损
5、伤） Block（阻碍） Transcription（转录） an outcome directly related to gene length. This has elicited（引出） the development of a dedicated repair system, transcription-coupled repair (TCR), which displaces or removes the stalled RNA polymerase and assures high priority repair. TRANSCRIPTIONAL STRESS, arising f
6、rom persistent blockage of RNA synthesis, constitutes an efficient trigger for p53-dependent apoptosis, which may be a significant anti-cancer mechanism. Lesions Also Interfere With DNA Replication（复制） Recently, a growing class of DNA polymerases（聚合酶）, numbered to , was discovered which seems devote
7、d specifically to overcoming damage-induced REPLICATIONAL STRESS. These special polymerases take over temporarily from the blocked replicative DNA polymerase-/, and possibly from pol . But this solution generally comes at the expense of a higher error rate. In fact, this process is responsible for m
8、ost of damage-induced point mutations and is thus particularly relevant for oncogenesis. Nevertheless, translesion polymerases still protect the genome. Double-strand DNA breaks (DSBs) induced by X-rays, chemicals or during replication of single-strand breaks (SSBs) and presumably during repair of i
9、nterstrand crosslinks are particularly relevant for the recombination machinery. Eg：Cells with specialized DNA recombination activities, such as B- and T-cells, may be very sensitive to DSBs when they are rearranging their immunoglobin or T-cell-receptor genes. This explains the frequent involvement
10、 of these genetic loci in oncogenic translocations in leukaemia(白血病) and lymphomas（淋巴瘤） and the preferential induction of these cancers by ionizing irradiation. Eg：DSBs also pose problems during mitosis（有丝分裂）, as intact（未受损的） chromosomes are a prerequisite（先决条件） for proper chromosome segregation（分离）
11、 during cell division. Thus, these lesions（损伤） frequently induce various sorts of chromosomal aberrations（染色体病）, including aneuploidy（）, deletions（缺失） (loss of heterozygosity, LOH) and chromosomal translocations（迁移，置换） events which are all intimately associated with carcinogenesis（癌变）.The cell-cycle
12、 machinery somehow senses genome injury and arrests（阻滞） at specific checkpoints in G1, S, G2 and M to allow repair of lesions before they are converted into permanent mutations. Lesion detection may occur by blocked transcription, replication or specialized sensors. When damage is too significant, a
13、 cell may opt for the ultimate mode of rescue by initiating（开始） apoptosis（凋亡） at the expense of a whole cell什么分子可作为DNA双链断裂/损伤的标志？用什么方法测定？ （1）Senescence(衰老), can be triggered when telomeres（端粒）the ends of linear chromosomescannot fulfil（执行） their normal protective functions. Here we show that senesce
14、nt human fibroblasts（纤维原细胞） display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show tha
15、t senescent cells contain activated forms of the DNA damage checkpoint kinases（激酶，致活酶） CHK1 and CHK2.（2）fluorescence（荧光）DNA发生双链断裂后最早反应之一是位于断裂点附近的组蛋白H2AX的C末端第139位丝氨酸残基被快速磷酸化形成-H2AX。磷酸化的-H2AX快速转导DNA损伤信号，导致下游分子磷酸化的激活，引发一系列的生物级联反应和和细胞学反应。-H2AX是迄今所研究的最重要的DNA损伤感应分子。免疫印记法检测。基于双链断裂/损伤，常在有丝分裂过程出现问题，引起各种类型染色体
16、畸变，包括非整倍体，缺失和染色体异位的发生，也用流式细胞仪做细胞倍体检查。采用PFGE法（脉冲场凝胶电泳法）测定链断裂量，以孔外进入凝胶的DNA占孔外和孔内总DNA比例FAR（DSB FAR（%）。用FITC-结合抗生素蛋白 检测DNA损伤。写出G1、G2/M checkpoint 基本信号通路主要分子成分。 G1-phase Checkpoint PathwayATM-CHK2-CDC25A-CDK2 axis forms a rapid response system;ATR-CHK1-CDC25A-CDK2;CDC25A-CDK4;ATM-CHK2/ATR-CHK1-P53-p21 pathway S-phase Checkpoin
