生物化学ppt课件DNA的生物合成和损伤修复DNA

Chapter 15DNA Biosynthesis and DNA Damage Repair Chapter 15 Section One The General Features of Replication of Chromosomal DNA Sectio生物化学ppt课件DNA的生物合成和损伤修复DNA1.DNA replicates semiconservatively.Watson and Crick predicted that DNA might semiconservatively replicate.DNA replicates semiconservativ the hypothesis _ p _ d _ p _ p _ d _ p the hypothes.In 1958 Meselson and Stahl demons-trated the semiconservative nature of DNA replication in E coli.In 1958 Meselson and Stahl The experiment:CsCl equilibrium gradient density ultracentri-fugation of 15N labeled E coli DNA.The exper The density of DNA was increased by labeling it with 15N,a heavy isotope of nitrogen.This was done by growing E coli 15 ge-nerations in a medium that contained 15NH4Cl as its only nitrogen source.The density of DNA was incre 15N DNA was extracted and subjected to CsCl equilibrium gradient density ultracen-trifugation.The DNA band position was recorded.There was one band of 15N DNA.15N DNA was extracted and sub The bacteria were transferred to an 14NH4Cl medium and grown for one generation.The DNA density was determined again.The position of the DNA band was compared with that of the 15N DNA.The bacteria were transferred What was the result?生物化学ppt课件DNA的生物合成和损伤修复DNA There was one DNA band.It had a lower density than 15N DNA because its position was above on that of 15N DNA.It was 15N/14N hybrid DNA.There was one DNA band.It ha After another generation growing in the 14NH4Cl medium the bacterial DNA density was determined.There were two DNA bands.After another generation grow One half of the DNA was 14N DNA,and another half was hybrid DNA.In succeeding generations the ratio of 14N DNA to hybrid DNA increased gradually.The hybrid DNA became less and less.One half of the DNA was 14N D summary DNA replicates in a semiconservative man-ner.When the two parental strands sepa-rate,each serves as the template for making a new,complementary strand.sum 2.The point at which separation of the strands and synthesis of new DNA takes place is known as the replication fork.The replication fork is Y-shaped.Two arms(V)are separated strands which act as the template and DNA synthesis is actively taking place.The body(I)is the parental DNA.2.The point at which separat 3.DNA replication is usually bidirectional.Replicon:Any piece which replicates as a single unit is called a replicon.All bacterial chromosomes and many phage and virus DNA molecules are circular and comprise single replicons.3.DNA replication is usually In contrast eukaryotic chromosomes consist of multiple replicons.Origin:The initiation within a replicon always occurs at a fixed point known as the origin.In contrast eukaryotic chromo.Terminus:In a circular replicon there is a single termination site roughly 180 opposite the unique origin.Terminus:In a circular rep Summary In a circular replicon replication begins from the fixed origin and forms two repli-cation forks.The two replication forks proceed bidirec-tionally away from the origin and the strands are copied as they separate until the terminus is reached.S 4.DNA replication is semidiscontinuous.The mechanism of DNA replication allows only for synthesis in a 53 direction.The two strands of DNA are antiparallel.4.DNA replication is semidis Question How is the parental strand that runs 53 past the replication fork copied?The answer is semidiscontinuous replica-tion.Quest At each replication fork one strand(the lead-ing strand),whose template runs 35 past the replication fork,is synthesized as one con-tinuous piece,while the other strand(the lag-ging strand),whose template runs 53 past the replication fork is made discontinuously as short fragments in the reverse direction.These short fragments are called Okazaki fragments.They are joined by DNA ligase and form the lagging strand.These short fragments are cal5.Origins contain short AT-rich repeat se-quences.Prokaryotic and eukaryotic origins have common features:a.They consist of multiple unique short repeat sequences.b.These sequences are recognition and binding sites of multi-subunit initiation factors.5.Origins contain short AT-ri c.These sequences are usually AT-rich.E coli s origin is called oriC.It is 254bp long and contains three 13-bp direct repeats and four 9-bp inverted re-peats.c.These sequences are usuall 6.DNA replication needs priming.DNA polymerases cannot initiate DNA replication by starting a new DNA chain.They can only add nucleotides to the 3 end of an existent piece of RNA or DNA under the direction of the template.The existent piece of RNA or DNA are called primer.6.DNA replication needs prim.The leading strand and all Okazaki frag-ments are primed by synthesis of a short piece of RNA(an RNA primer),which is then elongated with DNA by DNA poly-merase.There are also DNA priming or nucleotide priming.The leading strand and all 7.Multi-enzymes and proteins participate in DNA replication.Topoisomerases regulate the type and level of supercoiling of dsDNA.Helicases unwind the dsDNA.SSBs bind and stabilize the single DNA strand.Primase synthesizes the RNA primer.7.Multi-enzymes and proteins p.DNA polymerases elongate DNA chains.DNA ligase joins Okazaki fragments.生物化学ppt课件DNA的生物合成和损伤修复DNA8.DNA replication is of high fidelity.There are two types of replication errors.a.base(nucleotide)substitution.b.nucleotide insertion or deletion.There are two types of error controls a.presynthetic error control.b.proofreading control.mismatch repair.8.DNA replication is of high Section Two Features of DNA Polymerases 1.The substrates of DNA polymerases areThe substrates of DNA polymera 2.The active center of DNA pols catalyzes DNA synthesis.The active center can differentiate dNTP from NTP.DNA pols can choose the right nucleotide for base-pairing with the template nucleo-tide.2.The active center of DNA p3.The semi-closed right-handed structure of the DNA pol.is composed of three domains.thumb domain,fingers domain and palm domain.two active centers:polymerase active center and 3-5exonuclease active center.They are located in the palm domain.3.The semi-closed right-hande.The palm domain has three functions:a.polymerase activity b.to check the newly formed base pair c.proofreading control to remove the mis-paired nucleotide.The fingers domain binds the template strand and interacts with the nucleotide that enters the polymerase active center.The palm domain has three f.The thumb domain keeps the primer-template junction in position in the active center and makes the polymerase bind the substrates tightly.生物化学ppt课件DNA的生物合成和损伤修复DNA 4.The protein of sliding clamp,that encircles the DNA and interacts with the DNA pol,is responsible for the processivity of the DNA polymerase.Processivity of the DNA pol means DNA pol going on synthesis of DNA rapidly without stopping.4.The protein of sliding c Section Three DNA Replication in E coli生物化学ppt课件DNA的生物合成和损伤修复DNA1.E coli DNA replication initiates at oriC in a process mediated by a multi-protein com-plex.Protein factors participate in initiation at oriC include:DnaA,DnaB,DnaC,HU,to-poisomerase II(gyrase)and SSB.1.E coli DNA replication initi.DnaA protein forms a complex of 20-40 molecules,each bound to an ATP mole-cule,around which the oriC DNA with four 9-bp repeats becomes wrapped.This facilitates melting of three 13-bp repeats which open to allow binding of DnaB protein.DnaA protein forms a comple.With the help of DnaC,DnaB binds the opened DNA.DnaB is a helicase and can unwind dsDNA by using the energy of ATP hydrolysis.SSB binds the single DNA strand.Gyrase introduces negative supercoils into the dsDNA ahead of the replication fork.The prepriming complex is formed.With the help of DnaC,DnaB2.Primase synthesizes RNA primer.DnaG is a primase.It binds the template and is activated by DnaB.The activated primase synthesizes RNA primer.2.Primase synthesizes RNA pri3.DNA pol III elongates DNA and DNA pol I removes the primer.DNA pol III is the principal enzyme in elongation of DNA.The structure of the holoenzyme is composed of 10 different subunits in total number of 16.()22223.DNA pol III elongates DNA a Two core enzymes()2 are held together by a complex.subunit:DNA synthesisDNA synthesis subunit:proofreading subunit:the sliding clamp A single holoenzyme is responsible for the synthesis of both leading strand and Okazaki fragments of lagging strand.Two core enzymes()2 are The holoenzyme of DNA pol III has two core enzymes.One is responsible for the synthesis of leading strand.The other for the synthesis of Okazaki fragments.Because the template of the lagging strand is looped out both leading and lagging strand synthesis move in the same direction.The holoenzyme of DNA pol III When the lagging strand core enzyme completes an Okazaki fragment,it re-leases the strand.Then the primosome(DnaB-DnaG com-plex)synthesizes another primer and the core enzyme elongates and completes another Okazaki fragment.When the lagging strand cor.DNA pol I has only one polypeptide.It has three enzyme activities:a.the polymerase activity b.the 3 5 exonuclease activity c.the 5 3 exonuclease activity.Subtilicin can cut it into two fragments.DNA pol I has only one polThe large fragment is called klenow fragment.It has the polymerase activity and the 35 exonuclease activity.The 53 exonuclease removes the primer.The large fragment is called k The polymerase function simultaneously fills the gap with DNA by elongating the 3-end of the adjacent Okazaki fragment.The final phosphodiester bond between the fragments is made by DNA ligase.The polymerase function simul 4.Tus protein recognizes and binds to the TER site.That prevents the replication fork advancing.DNA replication terminates.4.Tus protein recognizes and 5.Only at the full-methylated oriC can initiate replication.There are 11 copies of sequence GATC in oriC.The dam methylase that recognizes the sequence GATC and places a methyl group on the A.5.Only at the full-methylatedGATC is a palindrome.The opposite strand also reads GATC in the 53 direction.5GATC3 3CTAG5Only at the full-methylated oriC can initiate replication.GATC is a palindrome.During DNA replication oriC is also re-plicated.The parental strand is methylated,but the newly synthesized daughter strand isnt.Although GATC in the daughter strand is also destined to become methylated,about 10 minutes elapse before this can happen.During DNA replication oriC is6.Two types of topoisomerases are required in DNA replication.DNA unwinding at the replication fork can generate positive supercoiling of the dsDNA ahead of the replication fork.DNA gyrase uses the energy of ATP hydrolysis to introduce negative su-percoiling into DNA hence removing supercoiling.6.Two types of topoisomerasesE coli chromosome is circular.When DNA replication is completed,there are two daughter circular DNA linked together(catenane).E coli topoisomerase IV can unlink the catenane.E coli chromosome is circular.Section Four DNA Replication in Eukaryotes Section Fou1.There are five types of common eukaryotic DNA polymerases:,.2.Eukaryotic and prokaryotic enzymes and protein factors that participate in DNA replication at the replication fork are comparable.There are five types of common3.After the polymerase/primase complex initiates replication polymerase starts elongation.4.There are two mechanisms of removing primers.a.RNase HI and FEN1-dependent mechanism.b.helicase Dna2 and EFN1-dependent mechanism.3.After the polymerase/prim5.The eukaryotic chromosome repli-cates only once in a cell cycle.a.pre-RC foprms in the G1-phase and is activated in the S-phase.b.Cdk controls the formation and activation of pre-RC.5.The eukaryotic chromosome r6.Telomerase participates in the replica-tion of telomere DNA.a.the problem of replicating the ends of linear chromosomes:The ends of linear chromosomes cant be fully replicated by semidiscontinuous replication as there is no DNA to elon-gate to replace the RNA removed from the 5-end of the lagging strand.6.Telomerase participates in Thus genetic information could be lost from the DNA.2.Telomere and telomerase solve the problem.To overcome this,the end of eukaryotic chromosomes(telomeres)consists of hundreds of copies of a simple non-informational repeat sequence(TTA GGG)with the 3-end overhanging the 5-end.Thus genetic information coulThe enzyme telomerase contains a short RNA molecule,part of whose sequence is complimentary to this repeat.This RNA acts as a template for the ad-dition of these repeats to the 3-over-hang by repeated cycles of elongation.The complimentary strand is then synthe-sized by normal lagging strand synthesis leaving a 3-overhang.The enzyme telomerase contains Section FiveDNA Replication in Mitochondria and Phages1.mtDNA replicates in D-loops.2.Phages circular DNA replicates in rolling circle.Secti Section Six The Repair of DNA Damage Section Six1.Physical or chemical agents may cause DNA damage.There are replication errors.There are several DNA repair systems in both prokaryotic and eukaryotic cells.1.Physical or chemical agents2.Mismatch repair system repairs the replication errors.Replication errors that escape proof-reading have a mismatch in the daug-hter strand.Hemi-methylation of the DNA after repli-cation allows the daughter strand to be distinguished from the parental strand.2.Mismatch repair system repaThe mismatched base is recognized and bound by MutS.MutS and DNA complex recruits MutL.MutH,an endonuclease,joins them and makes a nick in the newly synthesized DNA strand.The mismatched base is recogni Helicase UvrD and an exonuclease remove a piece of ssDNA that contains the error.DNA polymerase III fills the gap and DNA ligase seals the nick.Helicase UvrD and an exonucle 3.DNA Damage Repair Systems a.Direct Repair System The most common DNA damage is formation of thymine dimer by UV radiation.In the TT dimer there is a cyclobutane ring between the two neighbouring T residues in the same DNA strand.3.DNA Damage Repair SystemsThe photoreactivation repair system is a direct repair system.Direct repair can repair DNA damage without removing a base or nucleotide.In the photoreactivation repair the photolyase is activated by visible light and makes use of the light energy to break the cyclobutane ring.The TT dimer are restored to the original structure.The photoreactivation repair s b.Base Excision Repair SystemSingle base damages such as deamination of C,depurination,and depyrimidination are also very common.Glycosidases can recognize the damaged base and remove it.That results in an AP site(apurinic or apyrimidinic site).b.Base Excision Repair SyAP endonuclease hydrolyzes the phos-phordiester bond at the 5-end of the AP site.AP exonuclease cleaves the phosphor-diester bond at the 3-end of the AP site.and the deoxyribose-phosphate is re-moved.AP endonuclease hydrolyzes theThe gap left is filled with a nucleotide complementary to the template.DNA ligase makes the final phosphor-diester bond.The gap left is filled with a c.Nucleotide Excision Repair SystemNucleotide excision repair system recog-nizes the distortion of the DNA double helix.The distortion may be caused by TT,CT or CC dimer.c.Nucleotide Excision Repair In E coli the NER components consist of UvrA,UvrB,UvrC,and UvrD proteins.UvrA recognizes the distortion of DNAand combines with UvrB and ATP toseparate the dsDNA.UvrB recruits UvrC,an endonuclease.In E coli the NER components c UvrC makes nicks at both sides of the damage on the DNA strand.UvrD,a helicase,removes the damage contained fragment.DNA polymerase I fills the gap.DNA ligase links the 3-OH and 5-P by phosphordiester bond.UvrC makes nicks at both si Xeroderma Pigmentosum(XP)Human NER system consists of a numberof XP proteins including XPA,XPB,XPC,XPD,XPF,XPG and ERCC1.The complex of XP proteins scans the DNA,recognizes the lesion and removesa piece of damaged DNA about 25 nuc-leotides in length.Xeroderma PigmentosumThe gap is repaired by DNA polymeraseand DNA ligase.Patients having recessive mutations in XP protein genes cannot repair UV-induced DNA damages in the skin be-bause of lacking of XP protein activities.That causes cancer and is known asxeroderma pigmentosum.The gap is repaired by DNA pold.Recombinational repair(RR)system is responsible for repairing DNA double-strand breaks.The exchange of homologous regions between two DNA molecules is calledhomologous recombination(HR).d.Recombinational repair(RRHomologous recombination(HR)plays an important part in organisms.One of the HR functions is the repair ofDNA double-strand breaks.The hypothetical mechanism of RR is as follows:Homologous recombination(HR broken DNA5-33-5,5-33-5 intact homologous DNAThe broken region undergoes 5-exonuclease digestion.broken DNA5-3 5-33-5 3-55-33-5 unwinding and strand invasion 5-3 5-3 5-3 3-5 -5 5-3 -33-5 DNA synthesis5-3 5-5-33-5-53-55-3 resolution of Holiday junctions5-5-33-53-55-3生物化学ppt课件DNA的生物合成和损伤修复DNAIn E coli RecBCD protein complex hasboth nuclease and helicase activities.RecA protein participates in the formationof the Holliday junction.DNA polymerase synthesizes DNA.RuvA,RuvB,and RuvC proteins resolutethe Holliday junction.In E coli RecBCD protein comple.Translesional DNA polymerases can synthesize DNA translesionally.Translesional DNA pols require template for DNA synthesis,but they do not strictly obey the base-pairing rules in incorporation of nucleotides.Hence translesion DNA synthesis(TLS)is error-prone.A high mutation rate will occur.e.Translesional DNA polymerasIn E coli TLS is a part of a cellular stress response to extensive DNA damage known as SOS response.The SOS system involves a lot of genes which are related to DNA replication and repair.SOS system is regulated by LexA and RecA proteins.In E coli TLS is a part of a cLexA binds to the control region of SOS genes encoded for repair enzymes and shuts off the genes.Severe DNA damages activate the RecA mediated cleavage and destruction of LexA.That results in efficient expression of SOS response genes.LexA binds to the control regiDNA repair occurs with error-prone properties.Section Seven Reverse TranscriptionDNA repair occurs with error-p1.The discovery of reverse transcriptase develops the central dogma put forth by Crick.the central dogma replication replication DNA-RNA-protein transcription translationThe discovery of reverse transIn 1964 Temin put forth the hypothesis:virus RNA-provirus DNA-virus RNAIn 1970 Temin and Baltimore found the enzyme responsible for synthesis of DNA directed by RNA template in RNA virus independently.It is the reverse transcriptase.In 1964 Temin put forth the hy2.Reverse transcriptase makes use of RNA as template,tRNA as primer and synthesizes double-stranded cDNA.Reverse transcriptase has three enzy-matic activiyies:a.RDDP activity:It transcribes the RNA template onto a complimentary DNA strand to form an RNA-DNA hybrid.2.Reverse transcriptase makesRNase H activity:The enzyme then degrades the RNA template.DDDP activity:It replicates the resulting single-stranded DNA to form the duplex DNA.RNase H activity:The enzyme 3.The life cycle of retrovirusesRetroviruses have a diploid,positive sense RNA(mRNA)genome.The genome replicates via a dsDNA in-termediate,the provirus.The provirus is inserted into the host cells genome.3.The life cycle of retroviruThe provirus genome is expressible.The gene products are mRNA,the retrovirus genome,and various pro-teins,including reverse transcriptase.The retrovirus particle is packaged in the host cell and the mature virion buds from the host cell surface.The provirus genome is express Homework1.Explain the following terms:a.semiconservative replication b.replication fork c.leading strand d.lagging strand e.Okazaki fragment f.replicon g.primase h.telomere i.telomerase Homework k.mismatch repair l.photoreactivation m.replication origin answer the following questionsa.What are the substrates of the DNA polymerase during DNA synthesis?b.In what form are the deoxynucleotide units incorporated in the newly synthesized DNA c