DNA的生物合成与损伤修复-3

1、第十四-五章DNA biosynthesis and repairDNA的生物合成与损伤修复Central dogma of molecular biology中心法则双链互补反向Watson & Crick, Nature 1953理解DNA复制的基本原则DNA复制的关键调控元件DNA ReplicationDNA replication 基本特征Semi-conservative 半保留Origins of replication 复制起始点5-3 direction 53方向Bidirectional 双向Semi-discontinuous 半不连续RNA primers 复制引物Hi

2、gh fidelity 高保真DNA双链解螺旋分 别作为模板合成子链“Conserved”母链与 子链形成新的双螺旋子代保留了亲代全部遗传信息1. Semi-conservative其它方式1958年M. MeselsonF.W. Stahl2. Origins of replication复制起始点原核生物基因组为环形，有1个复制起始点真核生物基因组庞大，有若干复制起始点复制泡DNA strands open at the origin, forming 2 ReplicationForks (Y-shaped region)DNA在复制起始点解螺旋，形成2个复制叉（Y-形区）3. Bidi

3、rectional 一泡双向4. 5-3 direction, Why?energy4. 5-3 direction, Why?4. 5-3 direction, Proofreading碱基错配核酸外切酶去除加入正确碱基合成继续4. 5-3 direction, WHY?4. 5-3 direction, WHY?一个复制叉的两条互补链复制同时进行Leading strand 前导链沿5 3持续合成Lagging strand 后随链沿5 3合成 Okazaki fragments冈崎片段5. Semi-discontinuous 半不连续Replication fork6. RNA pri

4、mersGeorge W BushVs Chimpanzee99% sequence identityIdentity7. High fidelityGenetic diseasesMechanism of DNA replicationDNA拓扑异构酶DNA复制的酶学与拓扑学调控机制DNA引物酶DNA连接酶DNA聚合酶DNA聚合酶解旋酶单链结合蛋白TopoisomerasesHelicases PrimaseSingle strand binding proteins DNA polymerase Tethering proteinDNA ligasePrevents torsion by

5、DNA breaksseparates 2 strandsRNA primer synthesisprevent reannealing of single strandssynthesis of new strandstabilises polymeraseJoins adjacent DNA strands together (fixes “nicks”)Mechanism of DNA replication酶学与拓扑学调控机制Topoisomerase (Gyrase)拓扑异构酶Relieves stress caused by melting DNA减低DNA双螺旋打结、缠绕、连环C

6、leaves DNA and spins around itself to unwind helix切割 旋转Type I cleaves one strand, type II cleaves both strands拓扑酶I切割单链DNA，拓朴酶II切割双链DNAReseals DNA strands after relaxation重新链接DNA分子的磷酸二酯键解旋酶Hydrolyze ATP and opens up DNA helix水解ATP、解开DNA螺旋Moves 1000 bp/sec1000碱基/秒2 helicases: one on leading and one on

7、 lagging strand前导链和后随链各有一个SSB proteins aid helicase by destabilizing unwound ss conformation单链结合蛋白协助稳定单链DNA HelicaseSSBs help DNA helicase stabilizing ssDNA协助解旋酶维持DNA单链稳定状态Single strand binding protein (SSB)单链结合蛋白Creates a primer for DNA polymerase合成RNA引物Template-dependent 依赖DNA模板An RNA polymerase R

8、NA聚合酶Active briefly at beginning of strand synthesis在DNA单链合成初始阶段瞬时激活Primase引物酶DNA PolymeraseDNA聚合酶Enzyme that synthesizes a DNA strandUses existing strand as templateRequires a free “3 end” to add new nucleotidesHas several catalytic functionsSeveral forms existHigh Fidelity DNA ReplicationError rat

9、e= 1 mistake/109 nucleotidesAfforded by complementary base pairing and proof-reading capability of DNA polymeraseDNA PolymeraseDNA聚合酶缺口DNA连接酶连接2个DNA链末端5533RNA PrimerOkazaki FragmentDNA 聚合酶的5 - 3 核酸外切酶活性DNA聚合酶脱离DNAPol.5533RNA and DNA Fragments RNA PrimerDNAPol.3553RNA PrimerLigaseDNA ligaseDNA连接酶DNA

10、replicationDNA拓扑异构酶DNA引物酶DNA连接酶DNA聚合酶DNA聚合酶解旋酶单链结合蛋白Semi-conservative 半保留Origins of replication 复制起始点5-3 direction 53方向Bidirectional 双向Semi-discontinuous 半不连续RNA primers 复制引物High fidelity 高保真DNA replicationDNA replicationDNA ReplicationDNA ReplicationDNA Replication原核生物DNA Replication原核生物oriC (245bp

11、)单一复制起始点,oriCDNA解链, DnaA/DnaB 解旋酶/DnaC/拓扑异构酶/SSBPrimosome引发体：DnaB+DnaC+DnaG引物酶Replication forks move at 400-500 bp/secReplicate 4.6 x 106 bp in 40 minutes（245bp）for replication.2.The primosome (DnaB6 DnaG3) and DNA polymerase III holoenzymeDNA Replication原核生物1.the oriC is melted through DnaA, genera

12、ting single- stranded DNA substratesDNA Replication原核生物DNA聚合酶，不对称二聚体：一个作用于前导链另一个作用于随从链DNA Replication原核生物DNA聚合酶的小片段： 5-3外切酶DNA聚合酶的大片段： 3-5外切酶；5-3聚合酶DNA Replication真核生物Each eukaryotic chromosome is one linear DNA double helix线性，非环形Average 108 base pairs单个染色体太长With a replication rate of 2 kb/minute, r

13、eplicating one human chromosome would require 35days.Solution: DNA replication initiates at many different sites simultaneously.解决办法：多点起始DNA Replication真核生物DNA Replication真核生物原核生物复制的起始真核生物复制的起始双向复制 型复制双向复制多个复制单位(复制泡=复制起始点+复制叉)oriC多个起始点DnaA辨认起始点“蛋白质+DNA复合物”辨认起始点DnaA、B、C/拓扑异构 酶/SSB/引物酶DNA-pol、，PCNA ，拓

14、扑酶， 复制因子Eukaryotic DNA PolymerasesEnzymeLocationFunctionPol (alpha)NucleusDNA replicationincludes RNA primase activity, starts DNA strandPol (gamma)NucleusDNA replicationreplaces Pol to extend DNA strand, proofreadsPol (epsilon)NucleusDNA replicationsimilar to Pol , shown to be required by yeast mut

15、antsPol (beta)NucleusDNA repairPol (zeta)NucleusDNA repairPol (gamma)MitochondriaDNA replicationRNA primer near the end of chromosome on lagging strand cant be replaced with DNA since DNA polymerase must add to a primer sequence.后随链上染色质末端的RNA引物 不能置换成DNA，降解后造成 DNA缺失！遗传信息的丢失！？真核生物染色质末端复制终止Solution解决办法

16、Telomeres: Most eukaryotes have tandemly repeated sequences at the ends of their chromosomes.端粒：染色体末端的随机重复序列Telomerase (contains protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of new repeats.端粒酶：在染色体末端增加端粒长度 3.Compensates by len

17、gthening the chromosome. 维持或增加染色体长度4.Absence or mutation of telomerase activity can result inchromosome shortening and limited cell division.端粒酶缺失或突变导致染色体缩短，限制细胞分离Telomeres端粒Repeated G rich sequence on one strand in humans: (TTAGGG)n 富含G的重复序列Repeats can be several thousand base pairs long.In humans,

18、 telomeric repeats average 5-15 kilobases人细胞的端粒长达5-15KbTelomere specific proteins, eg. TRF1 & TRF2 bind to therepeat sequence and protect the ends端粒结合蛋白使以保护Without these proteins, telomeres are acted upon by DNA repair pathways leading to chromosomal fusions逃逸DNA损伤应答机制Structure of TelomeresElongated

19、 strand of telomere repeats are rich in guanine nucleotides5-TTTTGGGGTTTTGGGGTTTTGGGG-3They have the capacity to hydrogen bond to one another in the form of G-quartets.These create three-dimensional structures.Circulation April 19, 2011 vol. 123 no. 15 1650-1660TelomereTelomerase端粒酶Specialized rever

20、se transcriptase特殊逆转录酶Prevents “shortening ends problem” problem by adding telomeres to the end增加端粒长度Copies a small segment of RNA that it carries by itself自带RNA模板Requires a 3 end as a primer以3末端为引物Synthesis proceeds in 5 3 directionWhen active provides cell immortality持续激活造成细胞永生化5-CUAACCCUAAC-3Telo

21、meraseTERCTERTBlood. 2014 Oct 30;124(18):2775-83. doi: 10.1182/blood-2014-05-526285/bwindle/Telomerase/telomerase.htmlTelomerase and primaseTelomeraseReverse transcription逆转录依赖RNA的DNA聚合酶Reverse transcription逆转录A tRNA acts as a primer and hybridizes to a complementary part of the virus RNA genome cal

22、led PBS.Complementary DNA then binds to the U5 (non-coding region) and R region (a direct repeat found at both ends of the RNA molecule) of the viral RNA.A domain on the reverse transcriptase, RNAse H degrades the 5 end of the RNA which removes the U5 and R region.The “primer” “jumps” to the 3 end o

23、f the viral RNA and hybridizes to the complementary R region on the RNA.Reverse transcription逆转录The first strand of DNA (cDNA) is extended, and the majority of viral RNA is degraded by RNAse H.Second strand synthesis isinitiated from the viral RNA.There is then another jump where the PBS from the se

24、cond strand hybridizes with the complementary PB on the first strand.Both strands are extended further and can be incorporated into the hosts genome by integrase.Mitochondrial DNA replication线粒体DNA复制D-loopGenetic engineering基于DNA生物合成机制的基因工程Polymerase Chain Reaction DNA扩增DNA sequencing DNA测序Molecular

25、 cloning 分子克隆Reverse transcription 逆转录Gene editing 基因编辑1,000,000个DNA损伤/细胞/天碱基、核糖与磷酸二酯键容易损伤体内因素DNA复制错误：碱基错配、缺失、插入DNA自身不稳定：碱基丢失、碱基脱氨基活性氧：8-羟基脱氧鸟嘌呤体外因素物理因素：电离辐射（碱基氧化/脱落/DNA链交联断裂）、紫外线（嘧啶二聚体）化学因素：自由基、碱基类似物（5-溴尿嘧啶）、 碱基修饰剂、烷化剂、嵌入性染料生物因素：病毒DNA损伤类型碱基错配碱基缺失双链断裂单链断裂链间交联Intrastrand crosslink（链内交联） 嘧啶二聚体碱基修饰碱基缺失

26、化学修饰Photodamage光损伤DNA 损伤后的细胞命运Successful repair 成功修复Apoptosis 细胞凋亡Senescence 细胞衰老Transformation 细胞转化Tomas LindahlPaul ModrichAziz SancarAnnu Rev Physiol. 2013;75:645-68. doi: 10.1146/annurev-physiol-030212- 183715. Genome instability and aging.DNA repairDirect reversal of damage光复活修复嘧啶二聚体光复活酶Direct

27、reversal of damage烷基化碱基的直接修复O6-甲基鸟嘌呤- DNA甲基转移酶Base Excision Repair碱基切除修复Removal of the incorrect base by an appropriateDNA N-glycosylase to create an AP siteDNA糖基化酶水解去除已损伤碱基形成无碱基位点Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3-OH terminus adjacent to

28、the AP site AP核酸内切酶去除剩余核酸核糖部分Extension of the 3-OH terminus by a DNA polymerase, accompanied by excision of the AP siteDNA聚合酶以另一条链为模板合成互补序列Ligation by DNA ligaseDNA连接酶将切口重新连接Base Excision Repair碱基切除修复Base Excision Repair碱基切除修复Base Excision Repair碱基切除修复Human DNA GlycosylasesNucleotide Excision Repair

29、核苷酸切除修复Damage recognition损伤识别Double incision of the damaged strand on both the 5 and 3 sides 在损伤两侧切开，去除 一段寡核苷酸Filling in of the resulting gap by a DNA polymeraseDNA聚合酶介导新链合成Ligation连接Nucleotide Excision Repair核苷酸切除修复XPA-GMismatch Repair碱基错配修复MSH2:MSH6 complex binds the mismatch and identifies newly

30、synthesized strand.识别错配，区分新旧MLH1 endonuclease and other factors such as PMS2 bind, recruiting a helicase and exonuclease, which together remove several nucleotides including the lesion.切除错配片段The gap is filled by Pol and sealed by DNA ligase. 填空Mismatch Repair碱基错配修复调控MMR的蛋白因子Homologous Recombination同

31、源重组修复Homologous Recombination同源重组修复Homologous Recombination同源重组修复Non-homologous End Joining非同源末端连接Recombinational damage bypass重组跨损伤修复Model 1Model 2Translesion bypass synthesis跨损伤DNA合成Polymerase SwitchingDNA polymerasesHuman SyndromeMutatedGenePhenotypesDisrupted DNArepair pathwayAtaxia Telangiectas

32、ia (AT)共济失调-毛细血管扩张 症ATMCerebellar ataxia, telangiectases,oculomotor apraxia, predisposition to lymphoid malignancies, leukemias, immune defects, dilated blood vessel, infertility, metabolic defects, growth defectsDSB repair, DNAdamage signallingAtaxia Telangiectasia-likedisorder (A-TLD)MRE11Ataxia,

33、oculomotor apraxia,immunodeficiencyDSB repair, DNAdamage signallingNijmegan break syndrome(NBS)NBS1Microcephaly, immunodeficiency, growthdefects, mental retardation, B cell lymphoma, facial dysmorphismDSB repair, DNAdamage signallingNBS-like syndromeRAD50Microcephaly, facial dysmorphism, growthdefec

34、tsDSB repair, DNAdamage signallingRIDDLE syndromeRNF168Radiosensitivity,immunodeficiency,dysmorphic features and learningdifficultiesDSB repair, DNAdamagesignallingPrimarymicrocephaly 1MCPH1/BRIT1Microcephaly and mental retardationDSB repair, DNAdamage signallingHuman diseases and syndromes caused b

35、ydefective DNA repair人类DNA修复缺陷疾病Human SyndromeMutatedGenePhenotypesDisrupted DNArepair pathwaySeckel SyndromeATR, PCTN,SCKL2, SCKL3Severe intrauterine growth retardation,profound microcephaly, a bird-like facial profile, mental retardation and isolated skeletal abnormalities dysmorphic facial featur

36、esDSB repair, DNAdamage signallingRestrictive dermopathy(RD)LMNA,ZMPSTE24Tight adherent skin, joint contractures andrespiratory insufficiency, features of progeroid syndromes and premature death during gestationDSB repairHutchinson-Gilfordprogeria syndrome儿童早衰症LMNAProgeria (early growth retardation,

37、 shortstature, lipodystrophy, alopecia, stiff joints, osteolysis, dilated cardiomyopathy and atherosclerosis)DSB repair, DNAdamagesignallingLi-Fraumeni syndromeTP53Brain, breast cancer, sarcomas,leukemias,melanomas and gastrointestinal cancersDNA damagesignallingXeroderma Pigmentosum遗传性着色性干皮病XPA-XPG

38、, POLHSkin cancer, photosensitivity,neurodegeneration and microcephalyNERTrichothiodystrophyXPB, XPD,TTDANeurodegeneration, hypomyelination,progeria (cachexia, cataracts, osteoporosis), microcephaly, and psychomotoric abnormalities.NERHuman SyndromeMutatedGenePhenotypesDisrupted DNArepair pathwayCoc

39、kayne syndrome柯卡尼综合症CSA, CSB, XPB,XPD,XPGMicrocephaly, neurodegeneration,neuronal demyelination, microcephaly, progeria (skin atrophy, sparse hair cachexia, cataracts, hearing loss, retinopathy), photosensitivity, growth defectsNERCerebro-oculo-facio-skeletal (COFS) syndromeXPD, XPG, CSB,ERCC1Neuron

40、al demyelination anddysmyelination, brain calcification, microcephaly, neurodegeneration, progeria (cataracts, hearing loss, retinopathy), photosensitivity, growth defects and facial dysmorphismNERAtaxia with oculomotorapraxia 1 (AOA1)APTXAtaxia, neurodegeneration, oculomotorapraxia, hypercolesterol

41、emia and dysarthriaSSB repairSpinocerebeller ataxiawith axonal neuropathy(SCAN1)TDP1Ataxia, oculomotor apraxia, cerebellaratrophy, dysarthria, hypercholesterolemia,muscle weakness, sensory neuropathySSB repairImmunodeficiency withmicrocephalyXLFGrowth delay, recurrent infections,autoimmunity, hypogl

42、obulinemia, lymphopenia and microcephalyNHEJLigase IV syndromeLIG4Microcephaly, developmental and growthdelay, immunodeficiency (Aggamaglobulinemia, lymphopenia) and lymphoid malignanciesNHEJHuman SyndromeMutatedGenePhenotypesDisrupted DNArepair pathwayRadiosensitive severecombined immunodeficiency (RS-SCID)ARTEMISAggamaglobulinemia, lymphopenia, growthdefectsNHEJSevere combinedimmunodeficiency (SCID)RAG1,RAG2Aggamaglobulinemia, lymphopenia, growthdefectsNHEJBloom syndromeBLMMicro