分子生物学第三讲.ppt

单击此处编辑母版标题样式,,单击此处编辑母版文本样式,,第二级,,第三级,,第四级,,第五级,,,*,Molecular Biology,分子生物学,分子生物学,DNA复制,,DNA Replication,,类型,α – 螺旋 ( α –helix ),β－折叠 (β－pleated sheet),β－转角 (β－turn),,无规卷曲 (random coil),Ω环,蛋白质的二级结构是指多肽链主链的折叠和盘绕方式二级结构的基本单位,蛋白质的一级结构是指以肽键相连的肽链中氨基酸的排列顺序,Review of last class,,超二级结构是1973年Rossmann提出的，它是指二级结构的基本结构单位（α-螺旋，β-折叠等）相互聚集，形成有规律的二级结构的聚集体已知的超二级结构主要有,αα，βxβ，β-曲折和β-折叠筒,等βxβ：是两段平行的β-折叠通过一段连接链x连接而形成的结构如x为α- 螺旋则为βαβ；最常见的为两个βαβ聚集体连在一起形成βαβ αβ结构，称Rossmann折叠β-曲折（β-meander）：,是三条或三条以上反平行的β-折叠通过短链，如β-转角相连。

β-折叠筒（β-sheet barrel）：,,由多个β-折叠片卷成一个圆筒状的结构mRNA：,messenger RNA 信使RNA,,tRNA：,transfer RNA 转运RNA,,rRNA：,ribosomal RNA 核糖体RNA,RNA的种类,复性：,退火处理(慢速冷却)使得互补链互相配对,恢复双,,链结构,退火（ Annealing):,,DNA由单链复性变成双链结构的过程,,杂交（Hybridization):,,不同核酸链之间的互补部分的复性,,减色效应:,核酸复性时，紫外吸收降低，由于核酸复性而引起紫外吸收降低的现象,增色效应:,由于核酸变性而引起紫外吸收增加的现象Strong acid + high temperature:,completely hydrolyzed to bases, riboses/deoxyrobose(核糖/脱氧核糖), and phosphate,High pH (> 7-8) has subtle (small) effects on DNA structure,,High pH changes the tautomeric,(,互变异构),state of the bases.,RNA is unstable at higher pH,,（DNase）,,,（RNase）,,核酸外切酶,,,核酸内切酶,作用方式,底物,End of review,,,双脱氧链终止法,3’-TAGCACGATACTGTC-5’,5’-ATCGTGCTATGACAG-3’,,DNA复制,,DNA Replication,,教学要求:,1. 理解DNA复制的半保留机制和半不连续复制,,2. 掌握细菌DNA复制过程及有重要作用的酶和蛋白质,,3. 掌握真核生物DNA复制的特点,,4. 掌握DNA复制的类型,,DNA Replication: An Overview,Semi-conservative mechanism (,半保留机制,),,Replicons,(,复制子,), origins(,复制起始点,) and termini(,复制终点,),,semi-,discontinous,replication (,半不连续复制,),,RNA priming (RNA,引导,),,Semi-conservative mechanism (半保留机制),,DNA 双链解开，以单链做模板，碱基互补原则，各自合成一条子链,,During replication, the two parental strands separate and each acts as a template to direct the enzyme-catalyzed synthesis of a new complementary daughter strand following the normal base-pairing rules.,,,,,,,,亲代DNA,子代,子代,半保留复制,新合成,,的链,,15,N labeling experiment,15,N labeled DNA,unlabeled DNA,,,DNA半保留复制的生物学意义：,,DNA的半保留复制表明DNA在代谢上的稳定性，保证亲代的遗传信息稳定地传递给后代。

Replicon,,is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus.,,能进行复制的DNA 单位，从起始位点到终止位点的全部DNA.,Replicon 复制子,,Origin(复制起始位点),is the DNA sequence where a replicon initiates its replication.,A-T rich.,,Terminus(复制终止点),is the DNA sequence where a replicon usually stops its replication,,Prokaryotic genome 原核基因组:,single circular DNA = single replicon,,,Eukaryotic genome 真核基因组:,multiple linear chromosomes & multiple replicons on each chromosome,,细菌和真核生物复制的比较,,,,生物,复制子数,复制子长度,复制速率,细菌（,E. coli,）,1,4,200Kb,50Kb/min,酵母,,（,S. cerevisiae,）,500,40Kb,3.6 Kb/min,果蝇,,（,D. melanogaster,）,3,500,40Kb,2.6 Kb/min,爪蟾,,（,X. laevis,）,15,000,200Kb,500 bp/min,小鼠,,（,M. musculus,）,25,000,150Kb,2.2 Kb/min,植物,,（,V. faba,）,35,000,300Kb,,,Bidirectional,,replication of a circular bacterial replicon,,All prokaryotic(,原核的,) chromosomes and many,bacteriophage,and viral DNA molecules are circular and comprise single,replicon,.,,The origin,is a complex region,,where the initiation of DNA replication and the control of the growth cycle of the organism are regulated,,萤光素酶报告基因载体,,Bidirectional,replication (双向复制),,of a circular bacterial replicon,orign,terminal,daughter,,DNA,A single termination site is roughly 180,o,opposite the unique origin,,The long, linear DNA molecules of eukaryotic chromosomes consist of,mutiple,,replicons,, each with its own,orgin,.,,A typical mammalian cell has 50000-100000,replicons,with a size range of 40-200 kb.,,,When,,replication forks(,复制叉,),,from adjacent,,replication bubbles(,复制泡,),,meet, they fuse to form the completely replicated DNA.,Multiple,,eukaryotic replicons,,and replication bubbles,,replication forks(复制叉): 在复制启动时，尚未解开螺旋的亲代双链同新合成的两条子代双链DNA的交界处。

replication bubbles(复制泡)：两个靠得很近的复制叉之间形成的空间origins of DNA replication (every ~150 kb),replication bubble,daughter chromosomes,fusion of bubbles,bidirectional replication,Origins of DNA replication on mammalian chromosomes,5’,,3’,,3’,,5’,,5’,,3,’,3’,,5’,,3’,,5’,,5’,,3’,,,semi-discontinous replication (半不连续复制),5’,5’,3’,3’,5’,5’,3’,3’,5’,5’,3’,3’,Leading strand,Lagging strand,Okazaki fragment,半不连续复制,,,,,,半不连续复制概念,：,,,在DNA复制过程中，亲代DNA分子中以3’ 5’方向的母链作为模板指导新的链以5’ 3’ 方向连续合成，另一股以5’ 3’ 为方向的母链则指导新合成的链以 5’ 3’方向合成1000-2000个核 苷酸长度的许多不连续的片段（岗崎片段），,,这种复制方式称之为半不连续复制。

Leading strand（前导链）：,,,The leading strand is made continuously in a,5’ 3’direction from the origin.,,,DNA复制时，一股以3’ 5’方向的母链作为模板，指导新合成的链以5’ 3’方向连续合成的链称为前导链复制方向与解链方向一致,）,,Lagging strand（后随链):,,,The lagging strand is initiated at the replication fork and proceeds,5’ 3’ back towards the origin to form the first Okazaki fragment.,,,,DNA复制时，一股以5’ 3’方向的母链作为模板，指导新合成的链沿5’ 3’合成1000—2000个核苷酸不连续的小片段的链称为随从链，由岗崎等人在1968年发现复制方向与解链方向相反),,,又叫双螺旋稳定蛋白当解旋酶将双链打开以后，单链DNA具有一种潜在的恢复原来双链的能力，重新形成氢键而且单链本身若有反向重复也会形成发夹结构，这两种情况都会影响复制的，但SSB可以解决这一问题。

SSB并不是酶，是由177个aa组成的蛋白，,E. coli,的SSB以四聚存在，分子量为74KDa，结合在单链上，使DNA受到保护，不被酶水解，也不回复成双链，因此可以降低DNA的Tm值SSB对于细菌复制叉的结构的作用和解旋酶是同等的单链结合蛋白,,single strand binding protein, SSB,,岗崎片段（Okazaki fragment）：,,,,,DNA复制时，一股以5’ 3’方向的母链作为模板，指导新合成的链沿5’ 3’合成1000—2000个核苷酸不连续的小片段称之为岗崎片段岗崎片段由,DNA连接酶,连成一条完整的新链1、对酵母、细菌以及几种生活在真核生物细胞中的病毒来说，都可在DNA独特序列（ ）处观察到复制泡的形成复制起点,2、帮助DNA解旋的（ ）与单链DNA结合，使碱基仍可参与模板反应单链DNA结合蛋白(SSB),3、在DNA复制过程中，连续合成的子链称( )，另一条非连续合成,,的子链称为( )。

前导链,后随链,4、在DNA复制和修复过程中修补DNA螺旋上缺口的酶称为( )．,DNA连接酶,5、DNA后随链合成的起始要一段短的( )，它是由( )以核糖核苷酸为底物合成的RNA引物,DNA引发酶,6、在前导链上DNA沿5’→3’方向合成，在后随链上则沿3’→5’方向合成 （ ）,,DNA复制反应的共同特点,半保留复制,,半不连续复制,,有特定的复制起点,,需要引物,,复制真实性,,复制叉双向移动，,DNA,合成的单向延伸，,,,5’ 3’,,,,Proteins and enzymes of DNA replication,DNA,helicase,(DNA,解旋酶,),,single-strand binding protein (SSB,,单链结合蛋白,),,DNA,primase,(DNA,引发酶,),,DNA,topoisomerase,(,拓扑异构酶,),,,Primosome,(,引发体,),,DNA polymerase (DNA,聚合酶,),,DNA,ligase,(DNA,连接酶,),,DNA helicase (DNA解旋酶),Use the energy of ATP hydrolysis to move into and melt double-stranded DNA,,,利用ATP供能，解开DNA双链, 可随复制叉的伸展向前移动,,大肠杆菌中解旋酶的种类,种 类,功 能,DnaA,辨认起始点，并结合到复制起始部位,DnaB,解开DNA双链,DnaC,运送和协同DnaB,,DNA primase (DNA 引发酶),Synthesizes a short RNA primer to initiate synthesis of DNA,,,在模板复制的起始部位，以DNA为模板催化互补碱基聚合生成一小段RNA,,,e.g.,DNa G (,E. coli,),,,DNA topoisomerase (拓扑异构酶),Regulate the level of supercoiling of DNA molecules,,,Topoisomerase II: break both strands of DNA by the transfer of another double-stranded segment through the break.切断两条链而改变拓扑结构的酶。

如：存在于细菌中的DNA促旋酶、噬菌体T4的拓扑异构酶Ⅱ以及真核细胞中依赖ATP的拓扑异构酶Ⅱ等Topoisomerase I: break one strand of the DNA by passing the other strand through the break. 切断一条链而改变拓扑结构的酶，如：大肠杆菌的ω蛋白（ω-protein，由分子量11万的单个多肽链所成）及各种真核细胞中存在的切断-结合酶（nicking-closing enzyme，分子量约6万5千—7万的及分子量约10万的）核酸外切酶活性,3´→5´,外切酶活性：,,切除错配的核苷酸,5´→3´,外切酶活性：,,切除引物,,切除突变的片段,,,DNA ligase,,,make the final phosphodiester bond between the fragments,,连接DNA链3,,-OH末端和相邻DNA链5,,-P末端，形成磷酸二酯键，从而把两段相邻的DNA链连接成完整的链反应需要ATPATP,,DNA复制的方式,,1、,θ型复制,,2、滚环复制,,3、D环复制,,θ型复制 (Theta replication),在原核生物中发现的一种DNA复制机制。

复制是在环形DNA分子某一点(复制起始点)上开始的当复制围绕着染色体的两个方向进行时，所形成的复制泡状物延续生长是噬菌体中常见的DNA复制方式滚环复制的特点是以一条环状单链DNA为模板，进行新的DNA环状分子合成噬菌体的双链DNA环状分子先在一条单链的复制起点上产生一个切口(nick)，然后以另一条单链为模板不断地合成新的单链滚环复制(Rolling circle replication),释放出的新合成的单链或是先复制成双链DNA，被酶切割成单位长度后，再形成环状双链DNA分子；,或是释放出的新合成的单链DNA，先被酶切割成单位长度形成单链环状DNA分子后再复制成双链环状DNA分子滚环复制模型,1968年Gilbert提出：,,模板链和新合成的链分开,;,,不需,RNA,引物，在正链,3‘,－,OH,上延伸,,只有一个复制叉,;,,单向复制的特殊方式,,,,又称取代环复制双链在固定点解开进行复制，但两条链的合成高度不对称，一条链先复制，另一条链保持单链而被取代在电镜下看到呈英文字母D的形状D环复制 (D-loop replication),,Some researchers say an age-old cash crop long the focus of public health debate could be used to help solve the nation's energy crisis, by genetically modifying the tobacco leaf for use as a biofuel.,,碳中立,: 通过使用可再生能源，或者利用碳减排设施，使排放到空气中的二氧化碳总量和从大气中移除的二氧化碳总量达到平衡的实践。

HT: Herbicide tolerant,,,,Soybeans,,GM production area 2008: 65.8 million hectares,,share of production 2008: 70%A Romanian farmer shows genetically modified soybeans.,,Corn/Maize,,GM production area 2008: 37.3 million hectaresShare of production 2008: 24%A Mexican farmer cuts genetically modified corn in Capulalpan. Growing transgenic corn has been illegal in Mexico since 1998, but farmers say they received GM-corn from a government program.,,Cotton,,GM production area 2008: 15.5 million hectaresShare of production 2008: 46%A South African cotton farmer shows off a genetically engineered cotton plant. Some 90 percent of the 3,000 small-scale cotton farmers in the area use the insect-resistant Bt cotton variety. The GM cotton is resistant to the cotton bollworm pest, because it produces a naturally-occurring pesticide.,,Canola/Rapeseed GM production area 2008: 5.9 million hectaresShare of production 2008: 20%French farm workers leader and anti-GM activist Jose Bove cuts GM canola in a field near the town of Belpuech.Canola is fourth in the world league of GM crops. All genetically modified varieties are herbicide resistant. The crop is mainly used for biofuel production. Countries growing it commercially are Australia, the United States, and Canada.,,GM production worldwide 2008: experimental.Share of production 2008: n.a.Genetically modified Golden Rice is growing at the International Rice Research Institute (IRRI) in Los Banos, Manila. Golden Rice has been developed by Swiss scientists to fight Vitamin A deficiency. The rice contains a gene that allows the plant to produce beta-carotene, a precursor of Vitamin-A.,,Papaya GM production area 2008: 6,500 hectares.Share of production 2008: n.a.,,Greenpeace activists dump thousands of papayas in front of Thailand's Department of Agriculture in Bangkok. The protests were incited by the spread of illegal GM papaya in Thailand.,,Since 1998, GM papayas resistant to the ringspot virus have been cultivated in Hawaii and China. Critics say the protein used to make papayas virus-resistant is a potential allergen.,,Cowpea GM production worldwide 2008: experimentalShare of production 2008: n.a.A researcher in the biotechnology laboratory of the Nigeria-based International Institute of Tropical Agriculture (IITA) isolates genes from the protein-rich cowpea plant. Projects to further enhance their nutritional content are underway in Africa.So far, no major African food crop has received GM modification. Researchers at the IITA estimate, however, that biotechnology could help make Africa self-sufficient within 10 years.,,Alfalfa GM production area 2008: 100,000 hectaresShare of production 2008: n.a.A worker collects alfalfa at Cuatro Cienegas, Mexico. Alfalfa is mostly used as cattle feed in the form of dry hay.Herbicide tolerant GM alfalfa was commercialized in the United States from 2005 to 2007 but has been suspended pending an environmental impact statement by the U.S. Department of Agriculture.,，,,HoneySweet Plums,,GM production area 2008: experimentalShare of production 2008: n.a.GM plums grown in an experimental planting in the U.S. The plums are highly resistant to the Plum pox virus. Plum pox is not yet a problem in the U.S., but all plum varieties grown in the U.S. are susceptible to the virus. The U.S. Department of Agriculture considers this GM-variety as a last resort solution in case the Plum pox becomes endemic.,,Late Blight Resistant Potatoes GM production area 2008: experimentalShare of production 2008: n.a.Green tomatoes affected by late blight, which caused the Irish Potato Famine of the 1840s and 1850s. The fungus is killing potato and tomato plants in home gardens from Maine to Ohio and threatening commercial and organic farms.,,,,Farmers protest in front of Ministers' houses on T. Chowdaiah Road in Bangalore, demanding a ban on Bt Brinjal and other Bt crops, earlier this year.,,,。