核酸復(fù)制(雙語)

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?細(xì)胞增殖 細(xì)胞增殖的速度由細(xì)胞周期調(diào)控，成熟個(gè)體中的細(xì)胞按增殖狀態(tài)分為：1. 不增殖的終末分化細(xì)胞：神

4、經(jīng)元2. 穩(wěn)定型細(xì)胞：肝細(xì)胞、血管內(nèi)皮細(xì)胞3. 干細(xì)胞：胚胎干細(xì)胞、腫瘤干細(xì)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. 復(fù)制的基本規(guī)律 (Basic principles of DNA Replication)半保留復(fù)制: DNA復(fù)制過程中，新合成的兩個(gè)子代DNA分子與親代DNA分子的堿基順序完全一樣。每個(gè)子代DNA中的一條鏈來自親代DNA，另一條鏈?zhǔn)切潞铣傻?，這種合成方式稱為半保留復(fù)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.雙向復(fù)制: DNA復(fù)制過程中，從起始點(diǎn)(origin)向兩個(gè)方向解鏈，形成兩個(gè)延伸方向相反的復(fù)制叉(replication fork)，稱為雙向復(fù)

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.半不連續(xù)復(fù)制: 領(lǐng)頭鏈連續(xù)復(fù)制而隨從鏈不連續(xù)復(fù)制，就是復(fù)

13、制的半不連續(xù)性(semi-discontinuous replication)。 順著解鏈方向生成的子鏈，復(fù)制是連續(xù)進(jìn)行的，這股鏈稱為領(lǐng)頭鏈(leading strand)。 另一股鏈因?yàn)閺?fù)制方向與解鏈方向相反，不能順著解鏈方向連續(xù)延長，這股不連續(xù)復(fù)制的鏈稱為隨從鏈(lagging strand)。復(fù)制中的不連續(xù)片段稱為岡崎片段(okazaki fragment)。領(lǐng)頭鏈隨從鏈岡崎片段 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. 復(fù)制的酶學(xué) (Enzymology of DNA Replication)參與DNA復(fù)制的物質(zhì)底物(substrate): dATP, dGTP, dCTP, dTTP聚合酶(polymerase): 依賴DNA的DNA聚合酶，簡寫 為 DNA-pol模板(template) : 解開成單鏈的DNA母鏈引物(primer): 提

16、供3-OH末端使dNTP可以依次聚合 其他的酶和蛋白質(zhì)因子ATCG反應(yīng)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 不能使兩個(gè)dNTP直接聚合大腸桿菌DNA聚合酶：DNA pol/真核細(xì)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變的 DNA片段能辨認(rèn)錯(cuò)配的堿基對(duì)，并將其水解Prokar

17、yotes可能不可能可能基因突變后的致死性無無有多亞基不對(duì)稱二聚體？單肽鏈組成250120109分子量(kD)DNA-pol IIIDNA-pol IIDNA-pol I可能不可能可能基因突變后的致死性無無有53核酸外切酶活性多亞基不對(duì)稱二聚體？單肽鏈組成250120109分子量(kD)DNA-pol IIIDNA-pol IIDNA-pol I323個(gè)氨基酸小片段5 3核酸外切酶活性大片段/Klenow 片段 604個(gè)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 起始引發(fā)，有引物酶活性延長子鏈的主要酶，有解螺旋酶活性參與低保真度的復(fù)制 在起校讀、修復(fù)和填補(bǔ)缺口的作用在線粒體DNA復(fù)制中起催化作用DNA-pol DNA-pol DNA-pol DNA-pol Eukaryotes1、遵守嚴(yán)格的堿基配對(duì)規(guī)律2、聚合酶在復(fù)制延長中對(duì)堿基的選擇功能3、即時(shí)校讀功能Fidelity of DNA ReplicationProteins unwinding the helix in Prokaryotes理順DNA鏈拓?fù)洚悩?gòu)酶穩(wěn)定已解開的單鏈單鏈DNA結(jié)合蛋白SSB催化RNA引物生成引物酶DnaG (dnaG)運(yùn)送和協(xié)同DnaBDnaC (

20、dnaC)解開DNA雙鏈解螺旋酶DnaB (dnaB)辨認(rèn)起始點(diǎn)DnaA (dnaA)蛋白質(zhì)（基因）通用名功能 Primase1、5- 3聚合活性 催化子鏈5- 3方向核糖核苷酸之間通過磷酸二酯鍵連接，形成RNA引物；由于具有催化游離核苷酸之間通過磷酸二酯鍵連接的作用，起到引導(dǎo)子鏈起始合成的作用2、無外切活性DNA Topoisomerase1010 8 8 局部解鏈后局部解鏈后作用特點(diǎn)：酶切并合成DNA分子中磷酸二酯鍵拓?fù)洚悩?gòu)酶： 切斷DNA雙鏈的一股，形成松弛型環(huán)狀DNA。不需 ATP 供能拓?fù)洚悩?gòu)酶： 在無ATP時(shí)，切斷超螺旋DNA分子雙鏈中的某一部位，松弛超螺旋；在 ATP 供能時(shí)，松

21、弛型DNA變?yōu)樨?fù)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連接酶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復(fù)制過程 (Process of DNA Replication)DNA復(fù)制基本過程 (Basic Process of DNA Replication) Dna A Dna B、 Dna CDNA拓?fù)洚悩?gòu)酶引物酶SSB3535555RNA酶OHP5DNA-pol dNTP55PATP ADP+Pi55DNA連接酶原核與真核DNA復(fù)制的特異性 (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. 環(huán)狀DNA分子的復(fù)制是否會(huì)出現(xiàn)這個(gè)問題呢？ 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. 逆轉(zhuǎn)錄合成DNA (Reverse Transcription)北京大學(xué)醫(yī)學(xué)部的碩士生考試問答題： 鳥類Rous肉瘤病毒感染過程可被DNA合成抑制劑阻斷，其感染細(xì)胞過程并不被蛋白質(zhì)合成抑制劑阻斷這一現(xiàn)象說明了什么？其意義是什么？ 1970年，由于逆轉(zhuǎn)錄及RNA復(fù)制現(xiàn)象的發(fā)現(xiàn)，致使中心法則修訂如下：DNA RNA protein逆轉(zhuǎn)錄酶-RNA指導(dǎo)的DNA合成核糖核酸酶H-RNA的水解DNA聚

29、合酶-DNA指導(dǎo)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鼻咽癌宮頸癌獲得性免疫缺陷綜合征T細(xì)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損傷與修復(fù) (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. 光修復(fù) (light repair)2. 切除修復(fù) (excision repair)3. 重組修復(fù) (recombinant repair)4. SOS修復(fù) (SOS repair)Thymine dimerlight repairexcision re

39、pair著色性干皮病 (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