DNA的生物合成與損傷修復(fù)-2

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(bǔ)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(diǎn)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(diǎn)原核生物基因組為環(huán)形，有1個(gè)復(fù)制起始點(diǎn)真核生物基因組龐大，有若干復(fù)制起始點(diǎn)復(fù)制泡DNA strands open at the origin, forming 2 ReplicationForks (Y-shaped region)DNA在復(fù)制起始點(diǎn)解螺旋，形成2個(gè)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(cuò)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(gè)復(fù)制叉的兩條互補(bǔ)鏈復(fù)制同時(shí)進(jìn)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 p

4、rimersGeorge W Bush VsChimpanzee99% 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(jī)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 b

5、y 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(jī)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

6、環(huán)Cleaves 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(kāi)DNA螺旋Moves 1000 bp/sec1000堿基/秒2 helicases: one on leading and one

7、on lagging strand前導(dǎo)鏈和后隨鏈各有一個(gè)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(lài)DNA模板An RNA polymerase

8、 RNA聚合酶Active briefly at beginning of strand synthesis在DNA單鏈合成初始階段瞬時(shí)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 r

9、ate= 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(gè)DNA鏈末端5353RNA PrimerOkazaki FragmentRNA and DNA FragmentsDNA 聚合酶的5 - 3 核酸外切酶活性DNA聚合酶脫離DNAPol.5353RNA PrimerDNAPol.5353RNA 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(diǎn)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oriC (245bp)單一復(fù)制起始點(diǎn),oriCDNA解鏈

11、, 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 minutesDNA Replication原核生物（245bp）2.The primosome (DnaB6 DnaG3) and DNA polymerase III holoenzymeDNA Replication原核生物1.the oriC is melted through DnaA, generating single- st

12、randed DNA substrates for replication.DNA Replication原核生物DNA聚合酶，不對(duì)稱(chēng)二聚體：一個(gè)作用于前導(dǎo)鏈另一個(gè)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(xiàn)性，非環(huán)形Average 108 base pairs單個(gè)染色體太長(zhǎng)With a replication rate of 2 kb/minute,

13、 replicating one human chromosome would require 35 days.Solution: DNA replication initiates at many different sites simultaneously.解決辦法：多點(diǎn)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(gè)復(fù)制單位(復(fù)制泡=復(fù)制起始點(diǎn)+復(fù)制叉)oriC多個(gè)起始點(diǎn)DnaA辨認(rèn)起始點(diǎn)“蛋白質(zhì)+DNA復(fù)合物”辨認(rèn)起始點(diǎn)DnaA、B、C/拓?fù)洚悩?gòu)酶/SSB/引物酶DNA-pol、，PCNA

14、，拓?fù)涿?，?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 mu

15、tantsPol (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

16、法Telomeres: Most eukaryotes have tandemly repeatedsequences at the ends of their chromosomes.端粒：染色體末端的隨機(jī)重復(fù)序列Telomerase (contains protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeatand catalyzes the addition of new repeats.端粒酶：在染色體末端增加端粒長(zhǎng)度Compensates by lengthe

17、ning the chromosome.維持或增加染色體長(zhǎng)度Absence or mutation of telomerase activity can result in chromosome 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, telom

18、eric repeats average 5-15 kilobases人細(xì)胞的端粒長(zhǎng)達(dá)5-15KbTelomere specific proteins, eg. TRF1 & TRF2 bind to the repeat sequence and protect the ends端粒結(jié)合蛋白使以保護(hù)Without these proteins, telomeres are acted upon by DNA repair pathways leading to chromosomal fusions逃逸DNA損傷應(yīng)答機(jī)制Structure of TelomeresElongated stra

19、nd 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 reverse tr

20、anscriptase特殊逆轉(zhuǎn)錄酶Prevents “shortening ends problem” problem by adding telomeres to the end增加端粒長(zhǎng)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-3Telomeras

21、eTERCTERTBlood. 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(lài)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 called 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 w