核酸复制(双语)

1、DNA的生物合成 (Biosynthesis of DNA)DNA RNA proteinDNA ReplicationReverse Transcription A double stranded nucleic acid is duplicated to give identical copies. This process perpetuates the genetic information and keeps the daughter cells having almost the same genetic information as their parent cell. DNA

2、ReplicationDNA ReplicationWhy, When, and Where does the DNA replication occur?Why does the DNA replication occur?When does the DNA replication occur? The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. The stages are G1-S-G2-M. The G1 stage st

3、ands for GAP 1. The S stage stands for Synthesis. This is the stage when DNA replication occurs. The G2 stage stands for GAP 2. The M stage stands for mitosis, and is when nuclear and cytoplasmic division occur. Where does the DNA replication occur?细胞增殖 细胞增殖的速度由细胞周期调控，成熟个体中的细胞按增殖状态分为：1. 不增殖的终末分化细胞：神

4、经元2. 稳定型细胞：肝细胞、血管内皮细胞3. 干细胞：胚胎干细胞、肿瘤干细胞DNA ReplicationWhy: to maintain the genetic informationWhen: S phase of cell cycleWhere: in the nucleus of proliferating cellsIf something wrong with the process, so? Animals are multicellular organisms that require the normal function of all the organs of the

5、body. These organs are developed from different tissues and each of the tissues are products of cell division. For the body to function normally, the organs and tissues must communicate to control the development of the cells and tissues. Otherwise, uncontrolled cell growth in one part of the body c

6、ould induce diseases and even tumor. 复制的基本规律 (Basic principles of DNA Replication)半保留复制: DNA复制过程中，新合成的两个子代DNA分子与亲代DNA分子的碱基顺序完全一样。每个子代DNA中的一条链来自亲代DNA，另一条链是新合成的，这种合成方式称为半保留复制(semi-conservative replication)。 Conservative replication would leave intact the original DNA molecule and generate a completely

7、 new molecule. Dispersive replication would produce two DNA molecules with sections of both old and new DNA interspersed along each strand. Semiconservative replication would produce molecules with both old and new DNA, but each molecule would be composed of one old strand and one new one. In order

8、to determine which of these models was true, the following experiment was performed: The original DNA strand was labelled with the heavy isotope of nitrogen, N-15. This DNA was allowed to go through one round of replication with N-14, and then the mixture was centrifuged so that the heavier DNA woul

9、d form a band lower in the tube, and the intermediate (one N-15 strand and one N-14 strand) and light DNA (all N-14) would appear as a band higher in the tube. The replication is confirmed semiconservative. Each strand acts as a template for the synthesis of a new DNA molecule by the sequential addi

10、tion of complementary base pairs, thereby generating a new DNA strand that is the complementary sequence to the parental DNA. Each daughter DNA molecule ends up with one of the original strands and one newly synthesized strand.双向复制: DNA复制过程中，从起始点(origin)向两个方向解链，形成两个延伸方向相反的复制叉(replication fork)，称为双向复

11、制 (bidirectional replication) oriter The E.coli chromosome is a large, circular, double-stranded DNA molecule of 4600kb. The rate of synthesis is approximately 1000bp per second, the entire chromosome can be duplicated in about 38 minutes. Eukaryotic chromosomes are linear, double-stranded DNA molec

12、ules that are usually much larger than bacteria. Although the rate of fork movement in eukaryotes is slower than in bacteria, the presence of many independent origins of replication enables the larger genomes to be copied in the same amount of time as prokaryotic genomes.半不连续复制: 领头链连续复制而随从链不连续复制，就是复

13、制的半不连续性(semi-discontinuous replication)。 顺着解链方向生成的子链，复制是连续进行的，这股链称为领头链(leading strand)。 另一股链因为复制方向与解链方向相反，不能顺着解链方向连续延长，这股不连续复制的链称为随从链(lagging strand)。复制中的不连续片段称为岡崎片段(okazaki fragment)。领头链随从链冈崎片段 An Okazaki fragment is a relatively short fragment of DNA created on the lagging strand during DNA replic

14、ation. It was originally discovered in 1968 by Reiji Okazaki, Tsuneko Okazaki, and their colleagues while studying replication of Escherichia coli. When the lagging strand is being replicated, the 5-3 pattern must be used; Because of the need for DNA polymerase to synthesize in a 5to 3direction. Thu

15、s a small discontinuity occurs and an Okazaki Fragment forms. Finally, the fragments are ligated together to form a continuous strand. 复制的酶学 (Enzymology of DNA Replication)参与DNA复制的物质底物(substrate): dATP, dGTP, dCTP, dTTP聚合酶(polymerase): 依赖DNA的DNA聚合酶，简写 为 DNA-pol模板(template) : 解开成单链的DNA母链引物(primer): 提

16、供3-OH末端使dNTP可以依次聚合 其他的酶和蛋白质因子ATCG反应方程式(dNMP)n + dNTP - (dNMP)n+1 +PPiDNA polymeraseDNA聚合酶：5-3聚合活性 5-3外切活性 3-5外切活性 不能使两个dNTP直接聚合大肠杆菌DNA聚合酶：DNA pol/真核细胞DNA聚合酶：DNA pol/5 A G C T T C A G G A T A 3 | | | | | | | | | | |3 T C G A A G T C C T A G C G A C 5 3 5外切酶活性 5 3外切酶活性能切除突变的 DNA片段能辨认错配的碱基对，并将其水解Prokar

17、yotes可能不可能可能基因突变后的致死性无无有多亚基不对称二聚体？单肽链组成250120109分子量(kD)DNA-pol IIIDNA-pol IIDNA-pol I可能不可能可能基因突变后的致死性无无有53核酸外切酶活性多亚基不对称二聚体？单肽链组成250120109分子量(kD)DNA-pol IIIDNA-pol IIDNA-pol I323个氨基酸小片段5 3核酸外切酶活性大片段/Klenow 片段 604个氨基酸DNA聚合酶活性 3 5 核酸外切酶活性N 端C 端木瓜蛋白酶DNA-pol Klenow片段是研究中常用的工具酶 Exposure of DNA polymerase

18、I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5 - 3 exonuclease activity, and a large piece called Klenow fragment. The large or Klenow fragment of DNA polymerase I has DNA polymerase and 3 - 5 exonuclease activities, and is widely used in molecular biolo

19、gy. DNA-pol 起始引发，有引物酶活性延长子链的主要酶，有解螺旋酶活性参与低保真度的复制 在起校读、修复和填补缺口的作用在线粒体DNA复制中起催化作用DNA-pol DNA-pol DNA-pol DNA-pol Eukaryotes1、遵守严格的碱基配对规律2、聚合酶在复制延长中对碱基的选择功能3、即时校读功能Fidelity of DNA ReplicationProteins unwinding the helix in Prokaryotes理顺DNA链拓扑异构酶稳定已解开的单链单链DNA结合蛋白SSB催化RNA引物生成引物酶DnaG (dnaG)运送和协同DnaBDnaC (

20、dnaC)解开DNA双链解螺旋酶DnaB (dnaB)辨认起始点DnaA (dnaA)蛋白质（基因）通用名功能 Primase1、5- 3聚合活性 催化子链5- 3方向核糖核苷酸之间通过磷酸二酯键连接，形成RNA引物；由于具有催化游离核苷酸之间通过磷酸二酯键连接的作用，起到引导子链起始合成的作用2、无外切活性DNA Topoisomerase1010 8 8 局部解链后局部解链后作用特点：酶切并合成DNA分子中磷酸二酯键拓扑异构酶： 切断DNA双链的一股，形成松弛型环状DNA。不需 ATP 供能拓扑异构酶： 在无ATP时，切断超螺旋DNA分子双链中的某一部位，松弛超螺旋；在 ATP 供能时，松

21、弛型DNA变为负超螺旋。 DNA topoisomerases are a class of enzymes involved in the regulation of DNA supercoiling. Type I topoisomerases change the degree of supercoiling of DNA by causing single-strand breaks and re-ligation, whereas type II topoisomerases cause double-strand breaks. Both activities are especi

22、ally crucial during DNA transcription and replication, when the DNA helix must be unwound to allow proper function of large enzymatic machinery. DNA LigasePOO-O-OHO5POO-O-O335DNA连接酶ATPADP5353 DNA ligases catalyze formation of a phosphodiester bond between the 5 phosphate of one strand of DNA and the

23、 3 hydroxyl of the another. This enzyme is used to covalently link or ligate fragments of DNA together. The most widely used DNA ligase in laboratory is derived from the T4 bacteriophage. T4 DNA ligase requires ATP as a cofactor. A DNA ligase from E. coli is also available, but is not commonly used.

24、 The E. coli enzyme uses NAD+ as a cofactor. DNA复制过程 (Process of DNA Replication)DNA复制基本过程 (Basic Process of DNA Replication) Dna A Dna B、 Dna CDNA拓扑异构酶引物酶SSB3535555RNA酶OHP5DNA-pol dNTP55PATP ADP+Pi55DNA连接酶原核与真核DNA复制的特异性 (Specificity of DNA Replication between Prokaryotes and Eukaryotes )Initiation:

25、 Sequences of origin and RegulatorElongation: Length of Okazaki fragmentTermination: Replication of telomere DNA In eukaryotes, Okazaki fragments are typically a few hundred nucleotides long, whereas in prokaryotes they may contain several thousands of nucleotides. 环状DNA分子的复制是否会出现这个问题呢？ In human blo

26、od cells, the length of telomeres ranges from 8,000 base pairs at birth to 3,000 base pairs as people age and as low as 1,500 in elderly people. Cells normally can divide only about 50 to 70 times, with telomeres getting shorter until the cells become senescent, die or sustain genetic damage. Telome

27、res do not shorten with age in tissues such as heart muscle in which cells do not continually divide. Tumor can escape this fate by activating an enzyme called telomerase, which prevents the telomeres from getting even shorter. Measuring telomerase may be a new way to detect cancer. If scientists ca

28、n learn how to stop telomerase, they might be able to fight cancer by making cancer cells age and die. 逆转录合成DNA (Reverse Transcription)北京大学医学部的硕士生考试问答题： 鸟类Rous肉瘤病毒感染过程可被DNA合成抑制剂阻断，其感染细胞过程并不被蛋白质合成抑制剂阻断这一现象说明了什么？其意义是什么？ 1970年，由于逆转录及RNA复制现象的发现，致使中心法则修订如下：DNA RNA protein逆转录酶-RNA指导的DNA合成核糖核酸酶H-RNA的水解DNA聚

29、合酶-DNA指导的DNA合成Significance for the discovery of RT1. Broaden the central dogma 2. Challenge the central position of DNA 3. Applied for DNA engineering 4. Propose the virus-inducing tumor theoryHBV HPV HIV EB HTLV鼻咽癌宫颈癌获得性免疫缺陷综合征T细胞白血病肝癌 Reverse transcriptase is an enzyme thats part of the human immu

30、nodeficiency virus reading the sequence of viral RNA nucleic acids that have entered the host cell and transcribing the sequence into a complementary DNA sequence. Without reverse transcriptase, the viral genome couldnt become incorporated into the host cell, and couldnt reproduce. Reverse transcrip

31、tase sometimes makes mistakes reading the RNA sequence. So, viruses end up with a variety of subtle molecular differences in their surface coat and enzymes. Vaccines, which induce the production of antibodies that recognize and binding to very specific viral surface molecules, are an unlikely player

32、 in fighting HIV, because throughout infection, HIV surface molecules are continually changing.DNA损伤与修复 (Repair of Damaged DNA)DNA damage (mutation) A permanent change in the DNA sequence of a gene. How the damage occurs?1. Chemical and physical agents2. Biological agents3. Incorrect incorporation d

33、uring replication Mutations-heritable changes to the genetic material-are a fact life for all organisms. Despite the high degree of accuracy of DNA replication, and the numerous mechanisms to detect and repair errors, a strand of DNA can suffer permanent damage that is passed on to new generations o

34、f cells. Effects of such damage range from insignificant to devastating, from cancer or genetic defects in eukaryotes to adaptation and survival in bacteria.Significance of DNA mutation1. Molecular basis for life evolution 2. Change of genotype3. Death causing4. Molecular basis for disease With a mi

35、ssense mutation, the new nucleotide alters the codon so as to produce an altered amino acid in the protein product. With a nonsense mutation, the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (TAA, TAG, or TGA). Most amino acids are encoded by several differen

36、t codons. Silent mutations are said to be silent because they cause no change in their product.DNA mutation types1. Mismatch (Point mutation)2. Deletion3. Insertion4. Rearrangement (Inversion Duplication Translocation) Can DNA damage be passed from parents to their offsprings? If can, how the damage

37、 passes?DNA damage can be passed directly to the daughter cells following DNA replication and cell division. In multicellular organisms, mutations can be passed on to the next generation only if they occur in the germ line. Germ-line mutations may have no noticeable effect on the organism that conta

38、ins them but may have profound effects on progeny, especially if the mutated genes are important in development.Can DNA damage be corrected in vivo?Repair of damaged DNA1. 光修复 (light repair)2. 切除修复 (excision repair)3. 重组修复 (recombinant repair)4. SOS修复 (SOS repair)Thymine dimerlight repairexcision re

39、pair着色性干皮病 (xeroderma pigmentosis) Xeroderma pigmentosum (XP) was first described in 1874. In 1882, Kaposi coined the term xeroderma pigmentosum for the condition, referring to its characteristic dry, pigmented skin. XP is a rare disorder transmitted in an autosomal recessive manner. It is character

40、ized by photosensitivity, pigmentary changes, premature skin aging, and malignant tumor development. These manifestations are due to a cellular hypersensitivity to UV radiation resulting from a defect in DNA repair. recombinant repairSOS repair This years Nobel Prize awards discoveries of two viruse

41、s causing severe human diseases.The Nobel Prize in Phisiology or Medicine 2008 Harald zur Hausen, born 1936 in Germany, German citizen, MD at University of Dsseldorf, Germany. Professor emeritus and former Chairman and Scientific Director, German Cancer Research Centre, Heidelberg, Germany.Franoise Barr-Sinoussi, born 1947 in France, French citizen, PhD in virology, Institut Pasteur, Garches, France. Professor and Director, Regulation of Retroviral Infections Unit, Virology Department, Institut Pasteur, Paris, France.Luc Montagnier, born 1932 in France, French citizen, PhD in vi