上海师范大学-分子生物学5-DNA损伤修复及基因操作概述-kaigy课件.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,*,SECTION F,DNA损伤和修复,DNA damage,repair,link,F1,Mutagenesis,(诱变）,Mutation(突变),Replication fidelity（复制忠实性）,Mutagens:chemical&physical,Mutagenesis:direct&indirect,F1-1 Mutation突变,突变：DNA碱基水平上的,永久性,的，,可以遗传,的改变,Permanent,heritable,alterations in the base sequence of DNA,产生突变的原因,Reasons,DNA,复制或减数分裂重组过程中的自发性错误,Spontaneous errors,in DNA replication or meiotic recombination,由于理化试剂损伤,DNA,所,致,The,damaging effects of physical or chemical,mutagens,on DNA,转换,Transition,:嘌呤或嘧啶间的互换,Purine or pyrimidine is replaced by the other,如A,GT C,颠换,Transversion,:嘌呤与嘧啶间的互换,a purine is replaced by a pyrimidine or vice verse,如A,T or C,T,A or G,G,T or C,C,A or G,点突变,Point mutation,(单碱基改变,a,single base change,),DNA,非编码区,Noncoding,DNA,非,调节区,Nonregulatory,DNA,密码子的第三个碱基,3,rd,position of a,codon,沉默突变,Silent mutation,DNA编码区,改变了氨基酸,Coding DNA,altered AA,错义突变,Missense mutation,表型效应,Phenotypic effects,No,DNA编码区,产生新终止密码子,产生截短的蛋白,Coding DNA,stop codon,truncated protein,无义突变,Nonsense mutation,点突变的表型效应,Effects of a point mutation,Yes or No,Yes,插入或缺失,Insertions or deletions,移码突变Frameshift mutations,由于编码蛋白的基因被改变，结果导致所翻译出的蛋白从突变点到C末端都完全被改变了,The ORF of a protein encoded gene is changed so that the C-terminal side of the mutation is completely changed.,插入或缺失涉及一个或多个碱基的增加或丢失,The addition or loss of one or more bases in a DNA region,复制忠实性的分子机制,Molecular mechanisms for the,replication fidelity,只有当核苷酸与模板符合,Watson-,Crick,碱基正确配对原则时，,DNA,聚合酶才将其掺入,DNA,polymerase,:,Watson-Crick base pairing,聚合酶具有,3,5,外切酶活性，可去掉错碱基,3,5,proofreading,exonuclease,.,错配修复,Mismatch repair(F3),by,E.coli,polymerase,Proofreading,F1,Mutaagenesis,F1-3&4:突变 Mutagens,F1,Mutaagenesis,Mutation relevant,Cause DNA damage that can be converted to mutations.,物理诱变剂 Physical mutagens,高能离子化辐射：如X射线等可引起断链及碱基戊糖损伤,High-energy ionizing radiation,:X-rays and g-rays,strand breaks and base/sugar destruction,非离子化辐射如紫外线可引起嘧啶二聚体,Nonionizing radiation,:UV light,pyrimidine dimers,化学诱变剂 Chemical mutagens,碱基类似物,Base analogs,:direct mutagenesis,亚硝酸,Nitrous acid,:deaminates C to produce U,烷化剂,Alkylating agents,芳基化剂,Arylating agents,碱基类似物：正常碱基衍生物,Base analogs:,derivatives of the normal bases,incorporated in DNA,altering base pairing properties.,亚硝酸：使胞嘧啶脱氨成尿嘧啶，引起复制中发生,G,C,A,U,Nitrous acid,:,deaminates,C,to produce,U,resulting in G,C,A,U,F1-3&4:诱变 Mutagenesis,理化诱变剂所产生的绝大多数损伤，可在与复制叉相遇之前就被一种或多种修复机制所修复。,the great majority of lesions introduced by chemical and physical mutagens are,repaired,by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork,直接诱变 Direct mutagenesis,DNA中稳定的，具有改变了配对特性的未修复的碱基，在DNA复制中使得这种损伤（非永久性的）固定下来成为突变（永久可遗传的）。,The stable,unrepaired,base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication.,5-BrU,:G,:A,烯醇式enol form,Br,OH,H,O,Br,酮基异构体Keto form,H,O,AGCT,T,CCTA,TCGA,A,GGAT,AGCT,B,CCTA,TCGA,A,GGAT,Base analog,incorporation,AGCT,B,CCTA,TCGA,G,GGAT,AGCT,T,CCTA,TCGA,A,GGAT,1st round,of,replication,AGCT,B,CCTA,TCGA,A,GGAT,AGCT,C,CCTA,TCGA,G,GGAT,2nd round,of,replication,A,T,G,C,transition,5溴尿嘧啶,间接诱变 Indirect mutagenesis,转移损伤DNA合成,保持DNA的完整性但不保证序列的准确性,转移损伤DNA合成有时也称为“易错修复”,F2,DNA损伤,DNA damage,DNA损伤DNA lesions:,氧化性损伤oxidative damage,烷基化 Alkylation,聚化加和物 bulky adducts,DNA lesions,(DNA损害）,Oxidative damage,(氧化损伤）,Alkylation,(烷基化作用）,Bulky adducts,(加合物）,Occurs under Normal condition,Increased by,ionizing radiation,(physical,mutagens,),Alkylating agents,(Chemical mutagens),UV light,(physical mutagens),Carcinogen,(Chemical mutagens),F2-1DNA损伤,DNA lessions,损伤是DNA正常的化学或物理结构的改变。,An alteration to the normal,chemical,or,physical,structure of the DNA,The biological effect of the unrepaired DNA lesions,Lethal,(cell death),Physical distortion of,the local DNA structure,Blocks replication,And/or transcription,Mutagenic,Allowed to Remained in the DNA,A mutation could become fixed,by direct or indirect mutagenesis,Living cell,Altered chemistry of the bases,自发性DNA损伤,Spontaneous DNA lesions,Deamination,(,转氨作用）,:,C,U,methylcytosine,T,hard to be detected,Depurination,(,脱瞟呤作用,),:,break of the,glycosylic,bond,non-coding lesion,.,Depyrimidine,(,脱,嘧啶作用,),back,Chemical reactivity of bases is responsible for some DNA lesion,deamination,-ATG,C,TACG-,-TACGATGC-,-ATG,U,TACG-,-TACGATGC-,-ATG TACG-,-TACGATGC-,U,-ATG,C,TACG-,-TACGATGC-,Uracil DNA glycosylase,Cytosine deamination and repair,back,F2-2 氧化性损伤,Oxidative damage,氧化性损伤是活性氧分子如超氧化物及羟基自由基所产生的DNA损伤。,DNA lesions caused by,reactive oxygen species,such as superoxide and hydroxyl radicals,Oxidation products,在所以需氧细胞中，由于活性氧如超氧化物，氢过氧化物及羟基自由基的存在，在正常条件下会发生氧化损伤,occurs under NORMAL conditions in all aerobic cells,due to the presence of reactive oxygen species(ROS),such as,superoxide,hydrogen peroxide,and the hydroxyl radicals(,OH).,电离辐射引起的水辐射分解产生的羟基自由基能提高这些氧化产物的水平,The level of this damage can be INCREEASED,by hydroxyl radicals from the,radiolysis,of H,2,O caused by,ionizing radiation,F2-3 烷基化,Alkylation,烷化剂是可将烷基（如甲基）加入到核酸上各种位点的亲电化学试剂，如,甲基甲烷,磺酸盐和乙基亚硝基脲,Nucleotide modification caused by,electrophilic alkylating agents,such as methylmethane sulfonate,（MMS）,and ethylnitrosourea(ENU,),烷基碱基,亲电化学试剂加烷基到核酸上各种位点,Electrophilic,chemicals adds alkyl groups to various positions on nucleic acids,不同于正常甲基化酶的甲基化位点,Distinct from those,methylated,by normal,methylating,enzymes.,烷基剂,F2-4 聚化加合物,Bulky adducts,紫外线照射可形成嘧啶二聚体，使得DNA局部变性，破坏复制和转录。苯并芘代谢物可与鸟嘌呤共价结合成加合物，许多芳香烷化剂均可与DNA形成共价加合物。,DNA lesions that distort the double helix and cause localized denaturation,for example,pyrimidine dimers,and,arylating agents adducts,这些损伤破坏了DNA的正常功能。,These lesions,disrupt,the normal function of the DNA,Cyclobutane pyrimidine dimer,(嘧啶二聚体),Guanine adduct of,benzoapyrene,芳香烷化剂,共价加合物,back,F3,DNA修复 DNA repair,Photoreactivation,(光活化作用),Alkyltransferase,(烷基转移酶),Exision repair,(切割修复),Mismatch repair,(错配修复),Hereditary repair defects,(遗传修复缺陷),F3-1:光复活Photoreactivation,在可见光存在情况下，DNA光解酶可将环丁烷嘧啶二聚体再分解成单体,Monomerization of cyclobutane pyrimidine dimers by DNA,photolyases,in the presence of visible light,光复活是损伤被直接修复的例子，是无差错修复,Direct reversal of a lesion and is error-free,F3-2:烷基转移酶Alkyltransferase,烷基转移酶可以直接从突变的,O,6,-,烷基鸟嘌呤（可与T配对）上去除烷基。烷基转移到该蛋白并使之失活，故每个烷基转移酶只能用1次。,Removes the alkyl group from mutagenic,O,6,-alkylguanine which can base-pair with T.The alkyl group is transferred to the protein itself and inactivate it.,光复活是损伤被直接修复的例子，是无差错修复,Direct reversal of a lesion and is error-free,这种修复对烷化剂具有适应性：,E.coli,中低剂量烷化剂会诱发这一反应并提高了机体对高剂量的诱变效应和致死的保护能力。,The response is adaptive because it is induced in,E.coli,by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses,F3-3:切除修复 Excision repair,包括核苷酸切除修复和碱基切除修复两种形式,Includs,nucleotide excision repair(NER),and,base excision repair(BER),.,是,一种普遍存在的修复机制,Is a ubiquitous mechanism repairing a variety of lesions.,是无,差错修复,Error-free repair,Nucleotide excision repair,内切酶在损伤部位两边各切除精确数目的碱基,An,endonuclease,cleaves DNA a precise number of bases on both sides of the lesions(,UvrABC,endonulcease,removes,pyrimidine dimers,),包含损伤的,DNA,大,片段被切除移去,Excised lesion-DNA fragment is removed,缺口被填补和连接,The gap is filled by DNA,polymerase,I and sealed by,ligase,Base excision repair,DNA glycolases,cleaves apurinic or,pyrimidine site,DNA polymerase,DNA ligase,cleaves N-glycosylic bond,AP endonuclease,3,5 cleavage and,&,5,3 synthesis,F3-3:错配修复 Mismatch repair,错配修复是切除修复的一种特殊形式，用于修复在复制中错配并漏过校正检验的任何碱基。,A specialized form of,excision repair,which deals with any base,mispairs produced during replication,and which have escaped proofreading,F1,诱变,Mutagenesis,F2,损伤 DNA damage,Mutation:replication fidelity,mutagens,mutagenesis,F3,修复 DNA repair,DNA lesions:oxidative damage,alkylation,bulky adducts,Photoreaction,alkyltransferase,excision repair,mismatch repair,summary,Section G 基因操作 Gene manipulation,G1-1 DNA克隆 DNA cloning,G1-2,宿主和载体,Hosts and vectors,G1-3,亚克隆,Subcloning,G1-4,DNA文库,DNA libraries,G1-5,筛选文库,Screening libraries,G1-6,克隆分析,Analysis of a clone,基因操作 Gene,manipulation,G1,DNA,克隆概述,DNA cloning:an overview,基因操作 Gene,manipulation,基因克隆的重要环节就是把一个外源基因导入生物细胞，并使它得到扩增。,然而一个外源DNA片段是很难进入受体细胞的，即使进入细胞，一般也不能进行复制和功能的表达(,是因为,所得到的外源DNA片断一般不带有,复制子系统,及在新的受体细胞中进行功能表达的,调控系统,)。在基因工程中，通常是把外源DNA片断利用,运载工具,送入生物细胞。携带外源基因进入受体细胞的这种工具叫做,载体(Vector),G1-1 DNA cloning,(definition),DNA,克隆,定义,：将基因组中携有某一目的基因或其他相关序列的较小片段连接到一段可以自主复制的DNA（即载体）上，从而形成可以在另一宿主中进行复制的重组DNA，这种复制是独立于原初基因组的。带有重组DNA的宿主细胞的增殖构成了一群具有遗传一致性的个体（或称为单克隆)。这一系列操作过程就被称作DNA克隆。,DNA cloning is to place a relatively short fragment of a genome,which might contain the gene or other sequence of interest,in an autonomously replicating piece of DNA,known as a vector,forming,recombinant DNA,which can be replicates independently of the original genome,and normally in other host species altogether.Propagation of the,host organism,containing the recombinant DNA forms a set of genetically identical organism,or,a clone,.This process is called,DNA cloning,.,G1 DNA cloning:An Overview,遗传工程范畴,DNA序列分析，以及由此派生的蛋白质序列分析(参见J2)。,对基因启动子及其他调控序列的分离与分析(参见J4)。,通过大量正常和突变形式的产物研究来了解这些蛋白质/酶/RNA,的功能(参见J5)。,突变的鉴定，例如由于基因缺陷导致形成的疾病(参见J6)。,生物技术，如蛋白质及其他重要生物功能的分子如人胰岛素和,生长素的大规模工业化生产(参见J6)。,工程动物、工程植物以及基因治疗(参见J6)。,改变了特性的工程蛋白质(参见J6)。,G1-2,宿主和载体,Hosts and vectors,宿主细胞,Host organism/cell,:,where the plasmids get multiplied and propagated faithfully,which is crucial for DNA cloning.,用于,DNA,克隆的宿主,Hosts for DNA cloning vector,Prokaryotic host:,E.coli,(most cases),Eukaryotic host:Yeast,Saccharomyces cerevisiae,(large fragments of human genome),G1 DNA cloning:An Overview,载体具备的特性General features of a Vector,能够独立于宿主细胞基因组之外进行,自我复制,与分离,autonomously replicating DNA,independent of hosts genome,.,容,易,从宿主细胞中,分离,纯化,Easily to be isolated,from the host cell,大部分是环形的,Most are circular,some are linear,含有至少一个,选择标记,（,即某个基因能赋予宿主对某种毒素的抗性,，,或使宿主细胞能在特定生长条件下生存，从而能使含有该基因的载体的宿主可从那些不含该载体的细胞中被筛选出来,）,Contains at least one,selective marker,which allows host cells containing the vector to be selected amongst those which do not.,含有一个,多克隆位点,Contains a,multiple cloning site(MCS),G1 DNA cloning:An Overview,载体的类型Types of vectors,克隆载体,Cloning vectors,表达载体,Expression vectors,整合载体,Integration vectors,G1 DNA cloning:An Overview,克隆载体 Cloning vectors:,allowing the exogenous DNA to be inserted,stored,and manipulated at DNA level.,E.coli,cloning vector:plasmids,bacteriophages(l and M13),plasmid-bacteriophage l hybrids(cosmids).,Yeast cloning vector:yeast artificial chromosomes(YACs),MCS,表达载体 Expression vectors:,allowing the exogenous DNA to be inserted and expressed.,启动子和终止子是必需的,Promoter,and,terminator,for RNA transcription are required.,bacterial expression vectors,yeast expression vectors,mammalian expression vectors,G1 DNA cloning:An Overview,整合载体Integration vectors:,allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation.The integration is conducted by homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells.,细菌整合载体,bacterial integration vectors(,Agrobacterium,tumefaciens,Ti plasmid is used to integrate DNA into plant genome),酵母整合载体,yeast integration vectors,哺乳类整合载体,Mammalian integration vector:virus based,G1 DNA cloning:An Overview,G1-3,亚克隆,Subcloning,定义：将已克隆的DNA片段从一载体向另一载体的转移，这一过程被,称为,亚克隆,Transfer of a fragment of cloned DNA from one vector to another.,亚克隆的意义,亚克隆可用来对较大的克隆片段上较短区段进行仔细研究,Enables us to investigate a short region of a large cloned fragment in more detail.,可将基因转移到能在特定物种中表达的另一载体上,To transfer a gene from one plasmid to a vector designed to express it in a particular species.,G1 DNA cloning:An Overview,Preparation of plasmids containing a cloned DNA fragment(insert),Plasmid preparation,(vector),Restriction digestion,(trimming the DNA ends),Ligation,(join the insert and the vector),Transformation&selection of transformants,(introduce the plasmids into host cells),Assay of the recombinants,DNA Subcloning:a flow chart,Restriction,endonuclease,琼脂糖电泳Agrose Gel Electrophoresis:,Check your DNA at each step,Separation and Purification of DNA fragments of interests,Analysis of recombinant plasmids,ladder,Restriction analysis of a plasmid,G1 DNA cloning:An Overview,G1-4,DNA,文库,DNA libraries,DNA,文库定义：,DNA,文库是一套,DNA,克隆,，,每个克隆都是插入了不同片段的载体在宿主中扩增后的,产物。,DNA libraries,are sets of DNA clones,each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host.,一个克隆是一个遗传上独特的个体或一,群相同的个体。,A clone,is a genetically distinct individual or set of identical individuals,基因组文库和,cDNA,文库,Genomic libraries,and,cDNA,libraries,G1 DNA cloning:An Overview,基因组文库,是用基因组DNA的随机片段制备成的。然而用基因组文库来克隆某一基因是一种效率极低的方法，特别是对于庞大的真核生物,基因组，其中大量,DNA,是非编码的,Genomic libraries：,prepared form random fragments of genomic DNA,which may be inefficient to find a gene because of the huge abundance of the non-coding DNA,cDNA,文库是指,用来自表达目,的基因细胞或组织的,mRNA,作为来源构建成的文库。经反转录将mRNA合成cDNA(DNA拷贝)后，插入载体构建成cDNA文库。用DNA文库来克隆目的基因是有效的，但克隆的仅是其编码区，而不包含编码区以外的基因组序列。,cDNA libraries,：DNA copies(cDNA)synthesized from the mRNA by,reverse transcription,are inserted into a vector to form a cDNA library.Much more efficient in identifying a gene,but do not contain DNA coding functional RNA or noncoding sequence.,G1 DNA cloning:An Overview,G1-5,筛选文库,Screening libraries,通常筛选方法：是用一段与目的基因序列的某一区段互补或部分互补的放射性或荧光标记的,DNA,探针，通过杂交来检测出该基因，用,杂交来鉴定目的基因,Hybridization to identify the interested DNA or its RNA product,放射性探针,Radiolabeled,probes which is complementary to a region of the interested gene,探针,Probes:,根据蛋白产物序列推测出的一段寡核昔酸,An oligonucleotide derived from the sequence of a protein product of the gene,可以来自另一物种中的某一相关基因,A DNA fragment/oligo from a related gene of another species,点在膜,上,Blotting the DNA or RNA on a membrane,探针与,DNA,膜,杂交,Hybridize the labeled probe with DNA membrane(Southern)or RNA(Northern)membrane,找基因 Searching the genes of interest in a DNA library,G1 DNA cloning:An Overview,另一筛选方法是基于文库中被克隆的编码区表达后:,1)对其蛋白产物进行活性鉴定,2)或是用特异抗体对其进行鉴定,Identify the protein product of an interested gene,Protein activity,Western blotting using a specific antibody,G1 DNA cloning:An Overview,G1-6,克隆分析,Analysis of a clone,限制性酶切图谱,Restriction mapping,:,digestion of the with restriction enzymes.,测序,Sequencing,the cloned DNA,Table 1 须记忆 You must fully understand the function and application of all the enzymes listed in Table 1 before the final exam.,G1 DNA cloning:An Overview,G2,质粒,DNA,的制备,Preparation of plasmid DNA,G2-1,质粒载体,Plasmid as vectors,G2-2,质粒的小量制备,Plasmid minipreparation,G2-3,碱裂解,Alkaline lysis,G2-4,酚抽提,Phenol extraction,G2-5,乙醇沉淀,Ethanol precipitation,G2-6,氯化铯梯度,Cesium chloride gradient(purification),Gene manipulation,G2-1,质粒载体,Plasmid as vectors,质粒：质粒为染色体外的小型环状分子，大小约为2200kb，通常以多拷贝（可多达几百个）形式存在于宿主细胞大肠杆菌中。,Plasmids:,small,extrachromosomal circular molecules,from 2 to 200 kb in size,which exist in multiple copies within the host cells.,质粒含有复制起点，用以保证质粒的自主复制,Contain an,origin,of replication and replicate independently,质粒一般仅含携有几个基因,，,其中之一会赋予细菌对抗生素物质的抗性如,amp,抗性基因。,Usually carry a few,genes,one of which may confer resistance to antibacterial substance.,Example:,amp,抗性基因，编码可以降解青霉素类抗生素如氨苄青霉素的