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1、回顾第回顾第8章的内容章的内容n n相关的概念：半保留复制、半不连续复制、前导链、后随相关的概念：半保留复制、半不连续复制、前导链、后随相关的概念：半保留复制、半不连续复制、前导链、后随相关的概念：半保留复制、半不连续复制、前导链、后随链、冈崎片段、复制子、复制叉、链、冈崎片段、复制子、复制叉、链、冈崎片段、复制子、复制叉、链、冈崎片段、复制子、复制叉、 解旋酶、引物酶、拓扑解旋酶、引物酶、拓扑解旋酶、引物酶、拓扑解旋酶、引物酶、拓扑异构酶、异构酶、异构酶、异构酶、 DNADNA聚合酶、端粒酶、端粒。聚合酶、端粒酶、端粒。聚合酶、端粒酶、端粒。聚合酶、端粒酶、端粒。n nDNADNA复制的过程
2、复制的过程复制的过程复制的过程 ( (原核、真核原核、真核原核、真核原核、真核) )，包括起始，结合于解链，包括起始，结合于解链，包括起始，结合于解链，包括起始，结合于解链，延伸和终止。延伸和终止。延伸和终止。延伸和终止。n nDNADNA复制过程中涉及的酶或蛋白质有哪些？各自有何功能复制过程中涉及的酶或蛋白质有哪些？各自有何功能复制过程中涉及的酶或蛋白质有哪些？各自有何功能复制过程中涉及的酶或蛋白质有哪些？各自有何功能？n n原核生物与真核生物中参与原核生物与真核生物中参与原核生物与真核生物中参与原核生物与真核生物中参与DNADNA复制的复制的复制的复制的DNADNA聚合酶有哪些，聚合酶有哪
3、些，聚合酶有哪些，聚合酶有哪些，各自的活性及功能是什么？各自的活性及功能是什么？各自的活性及功能是什么？各自的活性及功能是什么？n nE.Coli E.Coli 基因组复制的过程？基因组复制的过程？基因组复制的过程？基因组复制的过程？n n端粒的结构如何，是如何复制的，端粒与衰老的关系如何端粒的结构如何，是如何复制的，端粒与衰老的关系如何端粒的结构如何，是如何复制的，端粒与衰老的关系如何端粒的结构如何，是如何复制的，端粒与衰老的关系如何？Chapter 9 The Mutability and Repair of DNAThree important sources for mutation
4、(unavoidable)1.Inaccuracy in DNA replication Inaccuracy in DNA replication (10(10-7-7 is not accurate enough) is not accurate enough)2.Chemical damage to the genetic materialChemical damage to the genetic material (environment)(environment)Lesions (arose from spontaneous damage) Lesions (arose from
5、spontaneous damage) Damage (caused by chemical agents and Damage (caused by chemical agents and radiationradiation3.Transposition Transposition ( chapter 11)( chapter 11) a class of Insertion generated by DNA a class of Insertion generated by DNA elements known as transposons)elements known as trans
6、posons)Chapter 9Errors in replication and damage to DNA have two consequences:1) Permanent changes to the DNA (mutations), which can alter the coding sequence of a gene or its regulatory sequences.2) Some chemical alterations to the DNA prevent its use as a template for replication and transcription
7、. Chapter 9Chapter 9To repair an error or damageFirst, Detect the errorsSecond, Mend/repair the errors or lesions in a way to restore the original DNA sequence.Chapter 9Chapter 9Questions to be addressed1.How is the DNA mended rapidly enough to prevent errors from becoming set in the genetic materia
8、l as mutation？2.How does the cell distinguish the parental strand from the daughter strand in repairing replication errors？3.How does the cell restore the proper DNA sequence when the original sequence can no longer be read?4.How does the cell deal with lesions that block replication? I. Replication
9、 errors and their repair （自发的）（自发的）II. DNA damage（外界的）（外界的）III. Repair of DNA damageOutlineChapter 9Chapter 9Chapter 9Chapter 91.How the replication errors are resulted?2.What is the nature of the replication errors? 3.How they are recognized and correctly repair?Section : Replication Errors and The
10、ir Repair1. Some Replication Errors Escape Proofreading Chapter 9Chapter 9 The 3-5 exonuclease component of replisome only improves the fidelity of DNA replication by a factor of about 100. But, thats not enough The misincorporated nucleotide needs to be detected and replaced, otherwise it will caus
11、e mutation.Figure 9-2 A mutation may be introduced by misincorporation of a base in the first round of replication. In the second round of replication, the mutation becomes permanently incorporated in the DNA sequence.Chapter 9Chapter 9The simplest mutations:（1）Transitions(转换转换): a kind of the simpl
12、est mutations which are pyrimidine-to-pyrimidine and purine-to-purine substitutions.（2）Transversions(颠换颠换): the other kind of mutation which are pyrimidine-to-purine and purine-to-pyrimidine substitutions.Chapter 9Chapter 92. The Nature of Mutations（3）Point mutations(点突变点突变): mutations that alter a
13、single nucleotide.（4）DNA microsatellites: Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, known as DNA microsatellites.are important in human genetics and disease；hard to be copied accurately and highly polymorphic (多态性多态性) in the population.Chap
14、ter 9Chapter 9（1）Insertions（2）Deletions （3）Gross rearrangements of chromosomeOther kinds of mutations (which cause more drastic changes in DNA)Chapter 9Chapter 9These mutations might be caused by insertion of transposon (转座子转座子)or by aberrant action of cellular recombination processes.Rate of sponta
15、neous mutation at any given site on chromosomal ranges from 10-6 to 10-11 per round of DNA replication, with some sites being “hotspot” . Chapter 9Chapter 9Figure 9-1 Base-change substitutionsChapter 9Chapter 9transitiontransversionmutation by base substration.swfATG,GTG,GAG,CCG,GCC,GAG,TAGATG,GTG,G
16、AG,CTG,GCC,GAG,TAGATG,GTG,GAG,CCG,GCC,GAG,TAGATG,GTG,GAG,CG,GCC,GAG,TAGATG,GTG,GAG,CGG,CCG,AGT,AGPoint mutationInsertions or deletionsChapter 9Chapter 9addition and deletion mutation 3. Mismatch Repair Removes Errors That Escape Proofreadingn nMismatch repair system: a system that increases the accu
17、racy of DNA synthesis by an additional two to three orders of magnitude（10-9-10-10）.n nThis system faces 2 challenges: a) rapidly find the mismatches/mispairs b) Accurately correct the mismatchChapter 9Chapter 9Important parts of mismatch repair systemFuctions of MutS:1. In E.Coli，MutS scans the DNA
18、, recognizes the mismatch from the distortion they cause in the DNA backbone. Chapter 9Chapter 92.MutS 2.MutS embracesembraces the the mismatch-containing mismatch-containing DNA, inducing a DNA, inducing a pronounced pronounced kinkkink in in the DNA and a the DNA and a conformational conformationa
19、l change change in MutS itself.in MutS itself.Functions of MutSImportant parts of mismatch repair systemChapter 9Chapter 9Chapter 9Chapter 9Figure 9-4 MutS is a dimer. One monomer interacts with the mismatch specifically, and the other nonspecifically. DNA is kinkedCrystal structure of MutS-DNA comp
20、lexBaseBackboneATPChapter 9Chapter 9Further steps of mismatch repair, we must pay attention to the other two important parts of the mismatch repair system-MutL and MutH.Chapter 9Chapter 9How these three parts interact?n nMutS-mismatch-containing DNA complex recruits MutL, MutL in turn activates MutH
21、, an enzyme causing an incision or nick on one strand near the site of the mismatch. n nNicking is followed by the specific helicase (Why?) and one of two exonucleases (Why?).Chapter 9Chapter 9n How does the E.coli mismatch repair system know which of the two mismatched nucleotides to replace?(Dam)
22、methylationChapter 9Chapter 9nDam methylase: The E.coli enzyme that methylases A residues on both strands of the sequence 5-GATC-3.（GATC广泛的分布于整个基因组中，约广泛的分布于整个基因组中，约256bp发生一次。）发生一次。）nThe newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis.(Dam) methylation
23、Chapter 9Chapter 9a. Replication generates hemimethylated DNA in E.coli.b. MutH makes incision in unmethylated daughter strand.Figure 9-5Dam methylase methylate A residues on both strands of 5-GATC-3Chapter 9Chapter 9Different exonucleases are used to remove single-strand DNA between the nick create
24、d by MutH and the mismatch.Figure 9-6Chapter 9Chapter 9Eukaryotic cellsn nEukaryotic cells also repair mismatches and do so using homologs to MutS (MSH) and MutL (MLH). The underlying mechanisms are not the same and not well understood. mismatch_repair.swfChapter 9Chapter 9Section : DNA Damage There
25、 are mainly three kinds of ways that DNA is damaged:1.DNA undergoes damage spontaneously (自发的自发的) from hydrolysis (水解水解) and deamination(转氨转氨)2.DNA is damaged by Alkylation (烷基化烷基化), Oxidation (氧化氧化) and Radiation (辐射辐射).3.Mutations are also caused by base analogs and intercalating (嵌入嵌入) agents.Cha
26、pter 9Chapter 91. Hydrolysis and DeaminationFigure 9-7Deamination CUHydrolysis creates apurinic deoxyribose Deamination 5-mC T Chapter 9Chapter 9nIf DNA naturally contained uracil instead of thymine, the deamination of cytosine will create a natrual base uracil which the repair system will not easil
27、y recognize.Can 5-mC T lesion be repaired?Why DNA contains T instead of U?Chapter 9Chapter 92. Alkylation, Oxidation and RadiationDNA is subject to attack from Reactive oxygen species (O2-, H2O2, OH)Figure 9-8 G modificationAlkylating chemical: Nitrosamines (亚硝胺亚硝胺)“O2-” hyperoxide“H2O2” Peroxide“OH
28、” hydroxylChapter 9Chapter 9UV(紫外线紫外线) induces a cyclobutane between adjacent thyminesFigure 9-9 Thymine dimer.相邻两个嘧啶之间发生光化学聚合相邻两个嘧啶之间发生光化学聚合, 形成胸腺嘧啶二聚体形成胸腺嘧啶二聚体(环丁烷环环丁烷环)。 nGamma radiation and X-rays (ionizing radiation) cause double-strand breaks and are particularly hazardous (hard to be repaired
29、).Chapter 9Chapter 93. Base analogs (碱基类似物碱基类似物) and intercalating agents (嵌入剂嵌入剂)Base analogs: similar enough to the normal bases to be processed by cells and incorporated into DNA during replication. n nBut they base pair differently, leading to mistake during replication.n nThe most mutagenic bas
30、e anolog is 5-bromouracil (5-BrU) (溴尿嘧啶溴尿嘧啶), an anolog of thymine.Chapter 9Chapter 9Chapter 9Chapter 95-bromouracil, base anolog of thymine, can mispair with guanineFigure 9-10a Base analogues烯醇异构体酮异构体A:T C:GChapter 9Chapter 9Intercalating agentsIntercalating agents are flat molecules containing se
31、veral polycyclic rings that interact with the normal bases in DNA through hydrogen bonds and base stacking.溴乙非啶二氨基吖啶/原黄素吖啶, 氮蒽Figure 9-10b Base analoguesChapter 9Chapter 9Section 3: Repair of DNA Damagen nSome damages, such as thymine dimer, nick or breaks in the DNA backbone, create impediments to
32、replication or transcriptionn nSome damages creates altered bases that have no effect on replication but cause mispairing, which in turn can be converted to mutation.Chapter 9Chapter 91.Direct reversal of DNA damage by photoreactivation (光激活作用光激活作用) and alkyltransferase (烷基转移酶烷基转移酶)2.Base excision r
33、epair (碱基切除修复碱基切除修复)3.Nucleotide excision repair4.Recombination (DSB) repairs5.Direct joining repairs6.Translesion DNA synthesisMechanisms to repair a damage1. Direct reversal of DNA damageChapter 9Chapter 9Figure 9-11 PhotoreactivationMonomerization of thymine dimers by DNA photolyases in the prese
34、nce of visible light.DNA光解酶光解酶 Chapter 9Chapter 9Direct reversal of DNA damageFigure 9-12 Methyl group removal.Methyltransferase catalyzes the transfer of the methyl group on O6-methylguanine to cystein residue (半胱氨酸半胱氨酸)on the enzyme, thereby restoring the normal G in DNA.direct_repair.swf2. Base E
35、xcision repair enzyme remove damaged bases by a base-flipping mechanismGlycosylase (糖基化酶糖基化酶) Chapter 9Chapter 9The damaged base which is flipped out The enzymeThe DNAFigure 9-14: base-flipping recognition by glycosylaseChapter 9Chapter 9Base excision repair systemsn nBase excision repair enzymesgly
36、cosylase(糖基化酶糖基化酶) recognize and remove damaged bases by a base-flipping mechanism, hydrolyzing the glycosidic bond.n nDNA glycosylases are lesion-specific.Chapter 9Chapter 9细胞中有多种具有不同特异性的细胞中有多种具有不同特异性的DNA糖基化酶。分别糖基化酶。分别可以识别并去除损伤碱基。目前已经知道有可以识别并去除损伤碱基。目前已经知道有8种不同种不同的的DNA糖基化酶糖基化酶。 1. The AP site is cre
37、ated by the hydrolysis of glycosylase bond.2. AP endonuclease & exonuclease cut out the 5 phosphate.3. DNA polymerase fill in the gap. Chapter 9Chapter 9Figure 9-13Chapter 9Chapter 9oxoG:A repair. A glycosylase recognizes the mispair and removes A. A fail-safe glycosylase also removes T from T:G mis
38、pairs, as if it knows how T is produced. Fail-safe systems (最后保险系统最后保险系统)Figure 9-15:防错糖基化酶防错糖基化酶 给给DNA糖基化酶糖基化酶除去修饰的碱基提除去修饰的碱基提供了第二次机会供了第二次机会 base excision repair.swfChapter 9Chapter 93. Nucleotide Excision repair enzymes cleave damaged DNA on either side of the lesionWhat is the difference between
39、the two kinds of excision repair systems?How does the nucleotide work?Chapter 9Chapter 91.Recognize distortions to the shape of the DNA double helix2.Remove a short single-stranded segment that includes the lesion. 3.DNA polymerase/ligase fill in the gap.与碱基切除修复不同，核苷酸切除修复酶不能区分各种不同的与碱基切除修复不同，核苷酸切除修复酶
40、不能区分各种不同的损伤，而是识别双螺旋形状上的扭曲。损伤，而是识别双螺旋形状上的扭曲。 Chapter 9Chapter 91) Once encountering a distortion UvrA exits the complex and UvrB melts the DNA to create a single-strand bubble around the lesion.2) Next, UvrB recruits UvrC, and UvrC creates two incisions in different positions on one strand.3) Finally
41、, DNA polymerase and ligase fill in the gap.Figure 9-16*nucleotide excision repair.swfChapter 9Chapter 9Nucleotide excision repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template Figure 9-17. Transcription-couple repair.核苷酸切除修复
42、不仅能修复整个基因核苷酸切除修复不仅能修复整个基因组中的损伤，而且当组中的损伤，而且当RNA聚合酶的转聚合酶的转录过程被某个基因发生转录链损伤终录过程被某个基因发生转录链损伤终止时，可进行转录偶联修复止时，可进行转录偶联修复（transcription-coupled repair）。）。此种修复主要是将核苷酸切除修复蛋此种修复主要是将核苷酸切除修复蛋白募集到受阻的白募集到受阻的RNA聚合酶上。聚合酶上。 Chapter 9Chapter 94. Recombination repairs DNA breaks by retrieving sequence information from u
43、ndamaged DNADouble-strand break (DSB) repair pathwayDetails are in chapter 10Chapter 9Chapter 9nThis is the very essential way that cells repair double-strand breaks in DNA in which both strands of the duplex are broken.nWe call it double-strand break (DSB) repair pathway, which retrieve sequence in
44、formation from sister chromosome.5. Direct joining repairsNHEJ: Nonhomlogous end joiningV(D)JChapter 9Chapter 96. Translesion DNA synthesis enables replication to proceed across DNA damageError- prone repair*Occurs when the above repairs are not efficient enough so that a replicating polymerase enco
45、unters a lesionTranslesion synthesis is also called a fail-safe or last resort mechanism. Chapter 9Chapter 9Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the damage site. Translesion polymerase is produced by cell in response to the
46、DNA damageTranslesion polymerases are expressed as part of the SOS response pathway. Chapter 9Chapter 9Crystal structure of a translesion polymerase.FIGURE 9-20 A Y-family polymerase found in many organisms. Chapter 9Chapter 9Translesion DNA synthesis in E. coliFIGURE 9-19 Translesion DNA synthesis
47、in E. coli 复制时一旦遇到损伤，复制时一旦遇到损伤，DNA聚合酶聚合酶III与其滑动夹与其滑动夹一起从一起从DNA上脱落，并被上脱落，并被移损移损DNA聚合酶所取代。聚合酶所取代。移损移损DNA聚合酶越过受损聚合酶越过受损部位（胸腺嘧啶二聚体）部位（胸腺嘧啶二聚体）进行进行DNA合成，然后移损合成，然后移损聚合酶被聚合酶被DNA聚合酶聚合酶III替替换。换。 DNA修复（修复（DNA repairing）是细胞对）是细胞对DNA受损伤后的一受损伤后的一种反应，这种反应可能使种反应，这种反应可能使DNA结构恢复原样，重新能执行结构恢复原样，重新能执行它原来的功能；但有时并非能完全消除它
48、原来的功能；但有时并非能完全消除DNA的损伤，只是的损伤，只是使细胞能够耐受着使细胞能够耐受着DNA的损伤而能继续生存。的损伤而能继续生存。虽然直接修复以及切除修复能够完全消除虽然直接修复以及切除修复能够完全消除DNA的损伤，而的损伤，而在实际中，在实际中，DNA经常发生双链断裂。如果对这种断裂不能经常发生双链断裂。如果对这种断裂不能进行及时的修复，就会使细胞产生致命性的后果。对于大进行及时的修复，就会使细胞产生致命性的后果。对于大多数细胞而言，用于修复双链断裂的主要机制就是同源重多数细胞而言，用于修复双链断裂的主要机制就是同源重组（组（Homologous Recombination)。部分
49、可以通过非同。部分可以通过非同源末端连接（源末端连接（NHEJ）的方式进行修复。）的方式进行修复。 Chapter 9Chapter 9Summary and key pointsAll the repair mechanisms (details) and the cause of the corresponding DNA errors and DNA damagesMismatch repair system: DNA replication errorsDirect reversal of DNA damages: utraviolet (UV) irradiation induced
50、 thymine/pyrimidine dimmers-photoactivation; alkylation agents caused O6-methylguanine-methyl transferase.Base excision repair: the base damage by alkylation and oxidation Nucleotide excision repair: the distortion of the DNA double helix by thymine dimmer or the bulky chemical adduct (加合物) on a bas
51、e.Recombination repair: double-strand breaks in DNA, errors encountered by a replication fork.Translesion synthesis allows the replication to proceed across DNA damage at a cost of error-prone replication. A different DNA polymerase is utilized.P272 表表9-1 DNA修复系统修复系统 熟记熟记 3.转座 Dam methylaseDam methy
52、lase思考题引起引起DNA突变和损失的原因有哪些？突变和损失的原因有哪些？o复制过程中的复制过程中的o环境因素（自发、物理、化学）环境因素（自发、物理、化学） 机体对于机体对于DNA突和变损失的修复机制突和变损失的修复机制有哪些，修复的主要机制是什么？有哪些，修复的主要机制是什么？(7种种=1+6)每种修复机制中设计涉及的关键蛋白或每种修复机制中设计涉及的关键蛋白或酶有哪些，他们的主要作用是什么？酶有哪些，他们的主要作用是什么？The causes of mutationvMutagensvError in replicationChemical PhysicalEnsuring the a
53、ccuracy Replication errors cause types Point mutation Insertions or deletionsDNA damage and their repairCausesRepairDirect reversal of DNA damageBase excision repair Nucleotide excision repairRecombination (DSB) repairsTranslesion DNA synthesisdamage spontaneouslyalkylation, oxidation and radiation base analogs intercalating agents诱变剂的种类 1.1.260260紫外辐射紫外辐射：环丁二聚：环丁二聚2.2.电离辐射电离辐射 3.3.加热加热:A-P:A-P位点位点 化学化学诱变剂诱变剂 1. 1.碱基类似物碱基类似物： 5-5-溴尿嘧啶溴尿嘧啶 2.2.脱氨剂脱氨剂：腺嘌呤：腺嘌呤次黄嘌呤次黄嘌呤 3.3.烷化剂：甲基磺酸乙脂烷化剂：甲基磺酸乙脂(EMS)(EMS) 4. 4.嵌入剂嵌入剂：EBEB物理物理诱变剂诱变剂
