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Chapter 3 Biosynthesis of DNA Replication Generation of a new copy of double-strand DNA from a parental DNA molecule.ReplicationParental DNADaughter DNASection I Basic rules of Replication.Semiconservative replication the fundamental feature.bidirectional replication.Semi-discontinuous replicationConservative modelSemiconservative modelRandom Dispersive modelParental DNA.Semiconservative replication is the fundamental feature.Experimental Evidence.DefinitionSemiconservative replication-DNA is synthesized by separation of the strands of a parental duplex,each then acting as a template for synthesis of a complementary strand.Each daughter molecule has one parental strand and one newly synthesized strand.A-TC-GT-AA-TG-C3 5 5 33 A C T A G5 5TGATC 3-T-G-A-T-C 5 3 G-A-T-C-A-5 3Parental DNADaughter DNAParentalstrand Daughter strand *The two parental strands separate and then each strand serves as a template;*4 kinds of dNTP as the substrate;*Catalyzed by DNA polymerase(DNA pol);*Follow the usual base-pairing rules of A with T and G with C;*Each daughter duplex has one parental strand and one new strand.Daughter DNAParental DNA.Significance Relative conservation of heredity The replication of genetic information in species from generation to generation assures the genetic continuity of that species.Genetic variations The long term survival of species depends on genetic variations that allow them to adapt to changing environments.bidirectional replicationOnce DNA replication is initiated at the origin(ORI),it continues in both directions around the DNA template.通过放射自显影观察通过放射自显影观察DNA的复制的复制 DNA双向复制的证据双向复制的证据 Figure.Circular DNA replication.The parental strands are shown in blue,and newly synthesized daughter strands are shown in red.The short segments represent the AT and GC base pairs connecting the complementary strands.Once DNA replication is initiated at the origin(ORI),it continues in both directions around the circular molecule until the advancing growing forks merge and two daughter molecules are produced.The origin is the only specific nucleotide sequence required for replication of the entire circular DNA molecule.v真核生物真核生物DNA复制也是从特定的起点开始双向进行复制也是从特定的起点开始双向进行的。然而与原核生物不同的是，在真核生物的的。然而与原核生物不同的是，在真核生物的DNA分子内有多个起始点，也就是说高等生物的染色体分子内有多个起始点，也就是说高等生物的染色体是多复制子的。是多复制子的。.Semi-discontinuous replication The two strands in the DNA duplex are opposite(5 3 and 3 5).Leading strand:The new strand that is continuously synthesized(in the same direction as replication fork movement).Lagging strand:The strand that is synthesized discontinuously in short pieces(Okazaki fragments)in a direction opposite to the direction of replication fork movement.Okazaki fragments:The Short segments of DNA formed in the discontinuous lagging strand synthesis of DNA and are rapidly joined by DNA ligase to form a continuous DNA strand.Section II Enzymology and topology of DNA Replication Substrate:dNTP(dATP,dGTP,dCTP,dTTP)Template:unwinding of DNA duplex Primer:RNA Enzyme:DNA-dependent DNA polymerase (DNA-pol or DDDP)Other enzymes and factors A T C A U G Subunits:dAMP dTMP dCMP dGMP 5end3endPhospho-diester bond HdNMP.Reaction (dNMP)n+dNTP(dNMP)n+1+PPi 5 3 direction.DNA-dependent DNA polymerase ()Characteristics of DNA-pol Substrate:dNTP polymerizing activity:5 3 direction The two strands in the DNA duplex are opposite(5 3 and 3 5)in direction,but all DNA polymerases catalyze nucleotide addition at the 3-hydroxyl end of a growing chain,so strands can grow only in the 5 3 direction.Template and primer directed enzyme It recognizes the next nucleotide on the DNA template and then adds a complementary nucleotide to the 3-OH of the primer or preexisting DNA.All DNA polymerases so far discovered can only elongate a preexisting DNA or RNA strand called the primer;they cannot initiate chains.Exonuclease activities:Removes nucleotides from an end of a polynucleotide strand.3 5 direction(proofreading)or 5 3 direction(excision)()Classification of DNA-pol1.DNA-pol in prokaryotes:DNA-pol I DNA-pol II DNA-pol IIIE.coliDNA-pol IDNA-pol IIDNA-pol III5 3 Polymerization +3 5 Exonuclease activity+5 3 Exonuclease activity+-Subunits 1110Molecules/cell4004020Function Repair enzyme Proofreading Timely removal of RNA primer Fill the gapunknownMajor DNA-synthesizing enzyme Properties of DNA PolymerasesDNA-pol Iprotease5 3 polymerizing activity5 3 exonuclease activity5 3 polymerizing activity3 5 exonuclease activity-proofreading functionN-terminalC-terminalProtease Small fragment323 A.A.5 3 exonuclease activityLarge fragment(Klenow fragment)604 A.A.5 3 polymerizing activity3 5 exonuclease activityTool enzyme used in molecular biology DNA Polymerases v尽管尽管DNApol是第一个被鉴定的是第一个被鉴定的DNA聚合酶，但聚合酶，但它不是在肠杆菌中它不是在肠杆菌中DNA复制的主要聚合酶。复制的主要聚合酶。主要证据如下主要证据如下:纯化的纯化的DNApol催化催化dNTP掺入的速率为掺入的速率为667碱基碱基/分，而分，而体内体内DNA合成速率要比此高二倍数量级合成速率要比此高二倍数量级;大肠杆菌的一个突变株中，此酶的活力正常，但染色体大肠杆菌的一个突变株中，此酶的活力正常，但染色体DNA复制不正常复制不正常;而在另一些突变株中，而在另一些突变株中，DNApol的活力中只是野生型的的活力中只是野生型的1%，但是，但是DNA复制却正常，而且此突变株增加了对紫外复制却正常，而且此突变株增加了对紫外线、烷化剂等突变因素的敏感性。线、烷化剂等突变因素的敏感性。v这表明该酶与这表明该酶与DNA复制关系不大，而在复制关系不大，而在DNA修复修复中中起着重要的作用。起着重要的作用。DNA-pol III:the major DNA-synthesizing enzyme 10 kinds of subunits form an unsymmetrical dimer Core polymerase:,subunits:sliding clamp protein -complex(other subunits):Promote holoenzyme assembly to the template and increase the core enzyme activity Core polymerase composed of,high polymerase activity()35 exonuclease()2.DNA-pol in eukaryotes:DNA-pol DNA-pol DNA-pol DNA-pol DNA-pol Mammalian Cells 5 3 polymeri-zation+3 5 Exonucle-ase activity-+Subunits 44422Cell location nucleinucleiMitochondrianucleinucleiFunction initiation strands Synthesis Primase activity RepairMitochondrialReplication Elongation strandsSynthesis Helicase activity Repair Proof-reading()Fidelity of replication is essential for the accurate transmission of genetic information Timely proofreading The 3 5 exonuclease activity of DNA-pol I and pol III detect and eliminate the occasional errors made by their polymerase function.DNA polymerase adds nucleotides to the 3 end of a strand of DNA.If a mismatch is accidentally incorporated,the polymerase is inhibited from further extension.Proofreading removes the mismatched nucleotide and extension continues.5 3 polymerizing activity3 5 exonuclease activity.Correct Watson-Crick base pairing DNA-pol III has the ability to select for incoming dNTP bases pairing with the template during replication.Core polymerase:,subunit:5 3 polymerizing activity subunit:The 3 5 exonuclease activity and function to select the dNTP for incorporation DNA 聚合酶之所以能正确地选择核苷酸参入到聚合酶之所以能正确地选择核苷酸参入到DNA 链链中去是通过酶的构象的改变来实现的。中去是通过酶的构象的改变来实现的。DNA 聚合酶反聚合酶反应分两个阶段进行应分两个阶段进行:(1)结合阶段，参入的结合阶段，参入的dNTP 与模板进行碱基配对，此时聚合与模板进行碱基配对，此时聚合酶处于酶处于开放的构象状态开放的构象状态不能催化聚合酶反应。不能催化聚合酶反应。(2)催化阶段催化阶段,新形成的碱基配对诱导聚合酶形成了新形成的碱基配对诱导聚合酶形成了闭合的构象闭合的构象从而正确地定位了催化基团的位置从而正确地定位了催化基团的位置(诱导契合诱导契合)。闭合构象的。闭合构象的形成是由参入的形成是由参入的dNTP 与模板正确的碱基配对诱导的，因此，与模板正确的碱基配对诱导的，因此，聚合酶的构象改变形成了对正确碱基配对的双重选择。聚合酶的构象改变形成了对正确碱基配对的双重选择。In addition,A set of DNA repair enzyme systems:Repairs errors in the newly synthesized DNA as well as any damage that may occur after its synthesis through chemical or physical insults.The inability of DNA polymerase to initiate chain elongation without a primer increases DNA replication fidelity.Cells maintain balanced levels of dNTPs.Enzymes for unwinding of DNA double strands Helicase（解螺旋酶）SSB（Single Stranded-DNA Binding protein,单链DNA结合蛋白）DNA topoisomerase（DNA拓扑异构酶)1.DNA Helicase（DNA解旋酶）解旋酶）Helicase in E.coli:DnaB protein Helicase in eukayotes:DNA-pol 2.SSB(single strand DNA binding protein)The separated strands are stabilized by DNA-binding proteins.Bind to ssDNA as a tetramer Keep the single strands apart Protect ssDNA segments from annealing and degrading by nucleases3.DNA topoisomerase Strand separation creates topological stress in the helical DNA structure,which is relieved by the action of topoisomerases,therefore these enzymes can prevent tangling of DNA strands and aid with the unwinding process.Partly unwoundChanges in topology of DNA More and more tensionSeparation of the two strands of DNA by helicase causes overwinding of the rest of the DNA molecule1)Type I topoisomerases cutting one strand of duplex DNA and then resealing the break not require ATPThere are two different classes of topoisomerases:DNA topoisomerase I action2)Type II topoisomerases change DNA topology by breaking and rejoining double-stranded DNA.cut both strands of DNA and reseal the cut utilizes ATP.DNA gyrase(Type II topoisomerases in prokaryotic cells)Inhibitors of Type II topoisomerases as antibiotics and anticancer chemotherapeutic agentsCiprofloxacin（环丙沙星）（环丙沙星）Novobiocin（新霉素）（新霉素）Doxorubicin（阿霉素）（阿霉素）Etoposide（依托泊苷）（依托泊苷）inhibit DNA gyrase but not eukaryotic topoisomerase II and are therefore antibioticsinhibit eukaryotic type II topoisomerase and are therefore widely used in cancer chemotherapy Primase Primase-DNA dependent RNA polymerase A specialized RNA polymerase that catalyzes the formation of RNA oligonucleotides used as primers for DNA synthesis.Primase does not proofread,primers are removed later,not inherited.Primase in prokaryotes:DnaG Primase in eukaryotes:DNA-pol DNA ligase DNA ligase catalyzes the formation of a phosphodiester bond between a 3 hydroxyl at the end of one DNA strand and a 5 phosphate at the end of another strand,thus joins adjacent completed fragments.Exerts function in:Replication,Repairing,Recombination,SplicingTool enzyme used in molecular biology Helicase:open hydrogen bonds and separate two strands using energy from ATP.Topoisomerase:cut DNA strand to release the tangle and reconnect them.SSB:bind to ssDNA to prevent it from degradation and anneal(rewind).Primase:synthesize short RNA primer(5 3)using DNA strand as template.DNA pol III:catalyze 4 dNTPs to become poly-deoxyribonucleotides(5 3)using DNA strand as template.DNA pol I:excise RNA primer and fill the gap with DNA fragment.DNA ligase:join the nick between two DNA fragments with phosphodiester bond Section III Biosynthesis of DNA Initiation Elongation Termination G1SG2MCell cycleDNA synthesis General Features of Replication Replication begins at a special site(replication origin),proceeds bidirectionally,and is semiconservative.DNA synthesis during replication occurs in the 5 to 3 direction.Replicon-A unit of DNA that is replicated from one replication origin.Bidirectional replicationReplication fork.Initiation1.Recognizing origin2.Unwinding DNA duplex3.Formation of primerThe arrangement of sequences in the E.coli replication origin,called oriC.Tandem repeats of three 13 bp sequences Four 9 bp inverted repeat sequences,binding sites for DnaA proteins.In eukaryotes,replication initiates at many dispersed sites along the DNA helixInverted repeats(Palindrome)-Reads the same backward as forward.For DNA,backward means on the other strand in the opposite physical direction.It does not mean backward on the same strand.Recognization of origin by DnaA to form initial complex HU:Histonelike protein stimulate initiation Unwinding of DNA double strand by DnaB DnaC helps DnaB SSB binding DnaA protein binds to the four 9bp inverted repeat sequences in oriC,forming an initial complex that contains 1020 protein subunits,initiates replication in E.coli.DnaB is an E.coli helicase that melts duplex DNADnaC helps DnaBSSB proteins:keep the two strands of DNA separated,and protect the DNA from nucleases cleaving.(2)Primosome and primerFormation of primer:RNA oligonucleotides used as for DNA synthesis.the 3OH.Primosome:The protein complex containing helicase(DnaB),DnaC,primase(DnaG),other factors and DNA initiation sequences that initiates synthesis of DNA.Formation of primerPrimer catalyzed by primase is a short stretch of RNA with a free hydroxyl group on the 3-end of the shorter strand.This hydroxyl group serves as the first acceptor of a nucleotide by action of DNA polymerase.Elongation()DNA synthesis proceeds in a 53 direction and is semidiscontinuous.One of the new DNA strands is synthesized continuously and the other discontinuously in short pieces,now called Okazaki fragments.The continuous or leading strand is the one in which 53 synthesis proceeds in the same direction as replication fork movement.The discontinuous or lagging strand is the one in which 53 synthesis proceeds in the direction opposite to the direction of fork movement.Leading strand-The strand that is continuously synthesized(in the same direction as replication fork movement).Lagging strand-The strand that is synthesized discontinuously in short pieces(Okazaki fragments)in a direction opposite to the direction of replication fork movement.The Okazaki fragments are then spliced together by DNA ligase.DNA-pol In eukaryotesDNA-pol In eukaryotesThe Leading and Lagging Strands Are Synthesized Concurrently in E.coliOkazaki fragments The Short segments of DNA(10002000 bases in bacteria,150-200 bases in eukaryotes)formed in the discontinuous lagging strand synthesis of DNA and are rapidly joined by DNA ligase to form a continuous DNA strand.Okazaki fragments are produced on the lagging strand as the result of discontinuous synthesisOn the lagging strand,DNA must loop to allow synthesisOkazaki Fragments are DNA-RNA hybrids.Termination of replication1.Remove RNA primer2.Fill the gap3.Join DNA fragments()Termination in prokaryotes Circular DNAoriter050SV403282oriterE.coli The Okazaki fragments are then spliced together by DNA ligase.DNA ligaseDNA polymeraseDNA polymerase I in prokaryotesRNAse H in eukaryotesRemove RNA primerFill the gapJoin DNA fragmentsDNA polymerase IDNA ligase Order of replication:Unwinding proteins.Single-strand binding proteins.Primase makes RNA primer.DNA polymerase makes DNA.Removing RNA primer(the RNA primer is removed by RNAse H in eukaryotes and DNA polymerase I in prokaryotes),DNA polymerase fills in gaps.DNA ligase joins gaps.Semiconservative replication,bidirectionally v Identification of the origins v Unwinding of dsDNA to get ssDNA template v Formation of the replication fork v Initiation of DNA synthesis and elongation Mechanism is similar to that in prokaryotes:.DNA biosynthesis in eukaryotesG1SG2MCell cycleDNA synthesisThe first step in DNA replication is the formation of the pre-initiation replication complex(the pre-RC).DNA replication in eukaryotes occurs only in the S phase of the cell cycle.Helicase:DNA-pol Primase:DNA-pol Topoisomerases Replication factor(RF):RFA,RFC RFA:SSB RFC:-complex PCNA clamps pol to the DNA PCNA:-clamp PCNA:proliferation cell nuclear antigen 增殖细胞核抗原 Initiation DNA-pol :Major synthesizing enzyme PCNA-interacts with pol -increases processivity DNA pol:proofreading Primers:more short(containing RNA or DNA)Okazaki fragments:more short Elongation 蛋白质蛋白质功功 能能RFA单链单链DNA结合蛋白，激活结合蛋白，激活DNA聚合酶，使解旋酶容易结聚合酶，使解旋酶容易结合合DNAPCNA激活激活DNA聚合酶和聚合酶和RFC ATPase活性活性RFC依赖依赖DNA的的ATPase，结合于引物，结合于引物-模板链，激活模板链，激活DNA聚聚合酶，促进合酶，促进PCNA结合于引物结合于引物-模板链模板链DNAPol/引物酶引物酶合成合成RNA-DNA引物引物DNA Pol/DNA复制，双螺旋解旋，核苷酸切除修复，校正错配碱复制，双螺旋解旋，核苷酸切除修复，校正错配碱基基FEN1核酸酶，切除核酸酶，切除RNA引物最后一个核苷酸引物最后一个核苷酸RNaseH1核酸酶，切除核酸酶，切除RNA引物引物DNA连接酶连接酶连接冈崎片段连接冈崎片段拓扑异构酶拓扑异构酶释放复制叉前进时产生的扭曲应力释放复制叉前进时产生的扭曲应力参与真核参与真核DNA复制的主要蛋白质复制的主要蛋白质 2.DNA复制过程复制过程 （1）引发体组装：）引发体组装：识别起点，局部解旋后，识别起点，局部解旋后，pol/引物酶复合引物酶复合物结合起始点物结合起始点DNA。由。由DNA解螺旋酶和解螺旋酶和pol/引物酶相互作用引物酶相互作用组装引发体。组装引发体。（2）合成引物：）合成引物：pol/引物酶在引物酶在RFA结合模板上合成结合模板上合成RNA引引物，并再延伸物，并再延伸DNA短序列（短序列（iDNA），形成），形成RNA-DNA引物。引物。（3）延伸，）延伸，pol/转换：转换：RFC结合引物结合引物3端，促进端，促进pol/引物引物酶去除。酶去除。PCNA结合结合DNA并引入并引入pol取代结合并活化。取代结合并活化。PCNA为为pol的活化因子。的活化因子。PCNA/pol复合物从引物复合物从引物3端持续延伸端持续延伸DNA链。合成前导链和随后链。链。合成前导链和随后链。（4）终止：）终止：由由RNA酶酶H1(一种对DNA-RNA杂合底物特异性杂合底物特异性的酶的酶)和和FEN1切除引物，切除引物，DNA连接酶封闭切口，形成连接酶封闭切口，形成2个完整个完整子代子代DNA。DNA ligaseDNA polymeraseRNase HRemove RNA primerFill the gapJoin DNA fragmentsDNA pol DNA ligase 真核生物复制的终止真核生物复制的终止Termination in eukaryotes Linear DNA Telomere3.Telomere and TelomeraseTelomere Telomeres are the structure at the end oflinear eukaryotic chromosome.Telomere are composed of tandem repeats of G rich DNA sequence and telomere associated protein.Telomeres are necessary for chromosome maintenance and stability.Functions:Form protective caps on the chromosome ends Necessary for chromosome maintenance and stability Telomere lengthTelomere length varies greatly between speciesTelomere Length Declines035651,5003,0008,000Age(years)Telomere length in base pairs(human white blood cells)长 短细胞分裂次数与端粒长短呈正比细胞分裂次数与端粒长短呈正比细胞分裂细胞分裂端粒阈值端粒阈值端粒长短端粒长短端粒酶活端粒酶活 Harley(1989)端粒的重复片段为探针检测端粒的重复片段为探针检测 胎儿细胞株胎儿细胞株婴儿细胞株婴儿细胞株青年细胞株青年细胞株老年细胞株老年细胞株年龄年龄小 大端粒长度端粒长度早老性侏儒症的端粒明显较正常人短早老性侏儒症的端粒明显较正常人短“多莉多莉”的衰老的衰老研究端粒（记时器）丢失的速率研究端粒（记时器）丢失的速率/年，预测人类的寿命年，预测人类的寿命 XX XY why?XX XY why?PCDPCD机制、癌细胞的无限繁殖机制、癌细胞的无限繁殖Telomerase An RNA-containing reverse transcriptase that using the RNA as a template,adds nucleotides to the 3 ending strand and thus prevents progressive shortening of eukaryotic linear DNA molecules during replication.Human telomerase RNA(hTR)Human telomerase associated protein 1(hTP1)Human telomerase reverse transcriptase(hTRT)TelomerasevTelomerase is a specialized reverse transcriptasevcontains both RNA and ProteinTelomeraseInchworn modelHow telomerase can solve the problem.In the first step,the primers are removed from the 5 ends of the daughter strands,leaving gaps.in the second step,telomerase adds extra telomeric DNA(red)to the 3 -ends of the other daughter strands.in the third step,DNA synthesis occurs,using the newly made telomeric DNA as a template.in the fourth step,the primers used in step three are removed.This leaves gaps,but the telomerase action has ensured that no net loss of DNA has occurred.Comparison of replication in prokaryotes and in eukaryotesProkaryoteEukaryoteEnzymeDNA-pol I,II,III,originsinglemultipleVelocity(bp/sec)2500(fast)250(slowly)Total Velocity equalPrimer 50-100 bp10-20 bpRemoving of primer DNA-pol IRNAse HOkazaki fragments1000-2000 bp100-200 bpSection Reverse Transcription1.reverse transcription Synthesis of a double-strand DNA from an RNA template.Retrovirus-A virus containing an RNA genome.Reverse transcriptase-A DNA polymerase that uses RNA as its template.3 activity:RNA-dependent DNA polymerase RNAse H DNA-dependent DNA polymerase 5-3 direction Primer:a kind of tRNA within the host cell Substrate:dNTPTemplate:virus RNA Useful tool in genetic engineeringReverse transcription in virusReverse transcription in vitrocDNAIntegration cDNAdNTPDNA-pol 3.D-loop replication:13 genes code for the ATP synthesis proteins 22 genes for tRNA 2 genes for rRNAmtDNA*The Central Dogma*Semiconservative replication *reverse transcription and Reverse transcriptase
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