DNA的生物合成與損傷修復(fù)-3

1、第十四-五章DNA biosynthesis and repairDNA的生物合成與損傷修復(fù)Central dogma of molecular biology中心法則雙鏈互補反向Watson & Crick, Nature 1953理解DNA復(fù)制的基本原則DNA復(fù)制的關(guān)鍵調(diào)控元件DNA ReplicationDNA replication 基本特征Semi-conservative 半保留Origins of replication 復(fù)制起始點5-3 direction 53方向Bidirectional 雙向Semi-discontinuous 半不連續(xù)RNA primers 復(fù)制引物Hi

2、gh fidelity 高保真DNA雙鏈解螺旋分 別作為模板合成子鏈“Conserved”母鏈與 子鏈形成新的雙螺旋子代保留了親代全部遺傳信息1. Semi-conservative其它方式1958年M. MeselsonF.W. Stahl2. Origins of replication復(fù)制起始點原核生物基因組為環(huán)形，有1個復(fù)制起始點真核生物基因組龐大，有若干復(fù)制起始點復(fù)制泡DNA strands open at the origin, forming 2 ReplicationForks (Y-shaped region)DNA在復(fù)制起始點解螺旋，形成2個復(fù)制叉（Y-形區(qū)）3. Bidi

3、rectional 一泡雙向4. 5-3 direction, Why?energy4. 5-3 direction, Why?4. 5-3 direction, Proofreading堿基錯配核酸外切酶去除加入正確堿基合成繼續(xù)4. 5-3 direction, WHY?4. 5-3 direction, WHY?一個復(fù)制叉的兩條互補鏈復(fù)制同時進行Leading strand 前導(dǎo)鏈沿5 3持續(xù)合成Lagging strand 后隨鏈沿5 3合成 Okazaki fragments岡崎片段5. Semi-discontinuous 半不連續(xù)Replication fork6. RNA pri

4、mersGeorge W BushVs Chimpanzee99% sequence identityIdentity7. High fidelityGenetic diseasesMechanism of DNA replicationDNA拓?fù)洚悩?gòu)酶DNA復(fù)制的酶學(xué)與拓?fù)鋵W(xué)調(diào)控機制DNA引物酶DNA連接酶DNA聚合酶DNA聚合酶解旋酶單鏈結(jié)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酶學(xué)與拓?fù)鋵W(xué)調(diào)控機制Topoisomerase (Gyrase)拓?fù)洚悩?gòu)酶Relieves stress caused by melting DNA減低DNA雙螺旋打結(jié)、纏繞、連環(huán)C

6、leaves DNA and spins around itself to unwind helix切割 旋轉(zhuǎn)Type I cleaves one strand, type II cleaves both strands拓?fù)涿窱切割單鏈DNA，拓樸酶II切割雙鏈DNAReseals DNA strands after relaxation重新鏈接DNA分子的磷酸二酯鍵解旋酶Hydrolyze ATP and opens up DNA helix水解ATP、解開DNA螺旋Moves 1000 bp/sec1000堿基/秒2 helicases: one on leading and one on

7、 lagging strand前導(dǎo)鏈和后隨鏈各有一個SSB proteins aid helicase by destabilizing unwound ss conformation單鏈結(jié)合蛋白協(xié)助穩(wěn)定單鏈DNA HelicaseSSBs help DNA helicase stabilizing ssDNA協(xié)助解旋酶維持DNA單鏈穩(wěn)定狀態(tài)Single strand binding protein (SSB)單鏈結(jié)合蛋白Creates a primer for DNA polymerase合成RNA引物Template-dependent 依賴DNA模板An RNA polymerase R

8、NA聚合酶Active briefly at beginning of strand synthesis在DNA單鏈合成初始階段瞬時激活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連接酶連接2個DNA鏈末端5533RNA PrimerOkazaki FragmentDNA 聚合酶的5 - 3 核酸外切酶活性DNA聚合酶脫離DNAPol.5533RNA and DNA Fragments RNA PrimerDNAPol.3553RNA PrimerLigaseDNA ligaseDNA連接酶DNA

10、replicationDNA拓?fù)洚悩?gòu)酶DNA引物酶DNA連接酶DNA聚合酶DNA聚合酶解旋酶單鏈結(jié)合蛋白Semi-conservative 半保留Origins of replication 復(fù)制起始點5-3 direction 53方向Bidirectional 雙向Semi-discontinuous 半不連續(xù)RNA primers 復(fù)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、)單一復(fù)制起始點,oriCDNA解鏈, DnaA/DnaB 解旋酶/DnaC/拓?fù)洚悩?gòu)酶/SSBPrimosome引發(fā)體：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，不對稱二聚體：一個作用于前導(dǎo)鏈另一個作用于隨從鏈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線性，非環(huán)形Average 108 base pairs單個染色體太長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.解決辦法：多點起始DNA Replication真核生物DNA Replication真核生物原核生物復(fù)制的起始真核生物復(fù)制的起始雙向復(fù)制 型復(fù)制雙向復(fù)制多個復(fù)制單位(復(fù)制泡=復(fù)制起始點+復(fù)制叉)oriC多個起始點DnaA辨認(rèn)起始點“蛋白質(zhì)+DNA復(fù)合物”辨認(rèn)起始點DnaA、B、C/拓?fù)洚悩?gòu) 酶/SSB/引物酶DNA-pol、，PCNA ，拓

14、撲酶， 復(fù)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.后隨鏈上染色質(zhì)末端的RNA引物 不能置換成DNA，降解后造成 DNA缺失！遺傳信息的丟失?。空婧松锶旧|(zhì)末端復(fù)制終止Solution解決辦法

16、Telomeres: Most eukaryotes have tandemly repeated sequences at the ends of their chromosomes.端粒：染色體末端的隨機重復(fù)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：在染色體末端增加端粒長度 3.Compensates by len

17、gthening the chromosome. 維持或增加染色體長度4.Absence or mutation of telomerase activity can result inchromosome shortening and limited cell division.端粒酶缺失或突變導(dǎo)致染色體縮短，限制細(xì)胞分離Telomeres端粒Repeated G rich sequence on one strand in humans: (TTAGGG)n 富含G的重復(fù)序列Repeats can be several thousand base pairs long.In humans,

18、 telomeric repeats average 5-15 kilobases人細(xì)胞的端粒長達5-15KbTelomere specific proteins, eg. TRF1 & TRF2 bind to therepeat sequence and protect the ends端粒結(jié)合蛋白使以保護Without these proteins, telomeres are acted upon by DNA repair pathways leading to chromosomal fusions逃逸DNA損傷應(yīng)答機制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特殊逆轉(zhuǎn)錄酶Prevents “shortening ends problem” problem by adding telomeres to the end增加端粒長度Copies a small segment of RNA that it carries by itself自帶RNA模板Requires a 3 end as a primer以3末端為引物Synthesis proceeds in 5 3 directionWhen active provides cell immortality持續(xù)激活造成細(xì)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逆轉(zhuǎn)錄依賴RNA的DNA聚合酶Reverse transcription逆轉(zhuǎn)錄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逆轉(zhuǎn)錄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線粒體DNA復(fù)制D-loopGenetic engineering基于DNA生物合成機制的基因工程Polymerase Chain Reaction DNA擴增DNA sequencing DNA測序Molecular

25、 cloning 分子克隆Reverse transcription 逆轉(zhuǎn)錄Gene editing 基因編輯1,000,000個DNA損傷/細(xì)胞/天堿基、核糖與磷酸二酯鍵容易損傷體內(nèi)因素DNA復(fù)制錯誤：堿基錯配、缺失、插入DNA自身不穩(wěn)定：堿基丟失、堿基脫氨基活性氧：8-羥基脫氧鳥嘌呤體外因素物理因素：電離輻射（堿基氧化/脫落/DNA鏈交聯(lián)斷裂）、紫外線（嘧啶二聚體）化學(xué)因素：自由基、堿基類似物（5-溴尿嘧啶）、 堿基修飾劑、烷化劑、嵌入性染料生物因素：病毒DNA損傷類型堿基錯配堿基缺失雙鏈斷裂單鏈斷裂鏈間交聯(lián)Intrastrand crosslink（鏈內(nèi)交聯(lián)） 嘧啶二聚體堿基修飾堿基缺失

26、化學(xué)修飾Photodamage光損傷DNA 損傷后的細(xì)胞命運Successful repair 成功修復(fù)Apoptosis 細(xì)胞凋亡Senescence 細(xì)胞衰老Transformation 細(xì)胞轉(zhuǎn)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光復(fù)活修復(fù)嘧啶二聚體光復(fù)活酶Direct

27、reversal of damage烷基化堿基的直接修復(fù)O6-甲基鳥嘌呤- DNA甲基轉(zhuǎn)移酶Base Excision Repair堿基切除修復(fù)Removal of the incorrect base by an appropriateDNA N-glycosylase to create an AP siteDNA糖基化酶水解去除已損傷堿基形成無堿基位點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核酸內(nèi)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聚合酶以另一條鏈為模板合成互補序列Ligation by DNA ligaseDNA連接酶將切口重新連接Base Excision Repair堿基切除修復(fù)Base Excision Repair堿基切除修復(fù)Base Excision Repair堿基切除修復(fù)Human DNA GlycosylasesNucleotide Excision Repair

29、核苷酸切除修復(fù)Damage recognition損傷識別Double incision of the damaged strand on both the 5 and 3 sides 在損傷兩側(cè)切開，去除 一段寡核苷酸Filling in of the resulting gap by a DNA polymeraseDNA聚合酶介導(dǎo)新鏈合成Ligation連接Nucleotide Excision Repair核苷酸切除修復(fù)XPA-GMismatch Repair堿基錯配修復(fù)MSH2:MSH6 complex binds the mismatch and identifies newly

30、synthesized strand.識別錯配，區(qū)分新舊MLH1 endonuclease and other factors such as PMS2 bind, recruiting a helicase and exonuclease, which together remove several nucleotides including the lesion.切除錯配片段The gap is filled by Pol and sealed by DNA ligase. 填空Mismatch Repair堿基錯配修復(fù)調(diào)控MMR的蛋白因子Homologous Recombination同

31、源重組修復(fù)Homologous Recombination同源重組修復(fù)Homologous Recombination同源重組修復(fù)Non-homologous End Joining非同源末端連接Recombinational damage bypass重組跨損傷修復(fù)Model 1Model 2Translesion bypass synthesis跨損傷DNA合成Polymerase SwitchingDNA polymerasesHuman SyndromeMutatedGenePhenotypesDisrupted DNArepair pathwayAtaxia Telangiectas

32、ia (AT)共濟失調(diào)-毛細(xì)血管擴張 癥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人類DNA修復(fù)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兒童早衰癥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遺傳性著色性干皮病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柯卡尼綜合癥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