生物化学课件DNA的生物合成和损伤修复DNA.ppt

1、 Chapter 15DNA Biosynthesis and DNA Damage Repair Section One The General Features of Replication of Chromosomal DNA1.DNA replicates semiconservatively.Watson and Crick predicted that DNA might semiconservatively replicate.the hypothesis _ p _ d _ p _ p _ d _ p .In 1958 Meselson and Stahl demons-tra

2、ted the semiconservative nature of DNA replication in E coli.The experiment:CsCl equilibrium gradient density ultracentri-fugation of 15N labeled E coli DNA.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 mediu

3、m that contained 15NH4Cl as its only nitrogen source.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.The bacteria were transferred to an 14NH4Cl medium and grown for one generation.The DNA

4、density was determined again.The position of the DNA band was compared with that of the 15N DNA.What was the result?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.After another generation growing in the 14NH4Cl m

5、edium the bacterial DNA density was determined.There were two DNA bands.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.summary DNA replicates in a semiconservative ma

6、n-ner.When the two parental strands sepa-rate,each serves as the template for making a new,complementary strand.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

7、 act as the template and DNA synthesis is actively taking place.The body(I)is the parental DNA.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 compri

8、se single replicons.In contrast eukaryotic chromosomes consist of multiple replicons.Origin:The initiation within a replicon always occurs at a fixed point known as the origin.Terminus:In a circular replicon there is a single termination site roughly 180 opposite the unique origin.Summary In a circu

9、lar 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.4.DNA replication is semidiscontinuous.The mechanism of DNA replica

10、tion allows only for synthesis in a 53 direction.The two strands of DNA are antiparallel.Question How is the parental strand that runs 53 past the replication fork copied?The answer is semidiscontinuous replica-tion.At each replication fork one strand(the lead-ing strand),whose template runs 35 past

11、 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 lig

12、ase and form the lagging strand.5.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.c.These sequences

13、 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.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 ex

14、istent piece of RNA or DNA under the direction of the template.The existent piece of RNA or DNA are called primer.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 primin

15、g or nucleotide priming.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.DNA polymerases elongate DNA chains.DNA

16、ligase joins Okazaki fragments.8.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.Section Two Featu

17、res of DNA Polymerases1.The substrates of DNA polymerases are 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.3.The semi-closed right-handed structure of

18、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.The palm domain has three functions:a.polymerase activity b.to check the newly formed base pair c.proo

19、freading 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 thumb domain keeps the primer-template junction in position in the active center and makes the polymerase bind the substrat

20、es tightly.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.Section Three DNA Replication in E coli1.E coli DNA r

21、eplication 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.DnaA protein forms a complex of 20-40 molecules,each bound to an ATP mole-cule,around which the oriC DNA with fo

22、ur 9-bp repeats becomes wrapped.This facilitates melting of three 13-bp repeats which open to allow binding of DnaB protein.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

23、negative supercoils into the dsDNA ahead of the replication fork.The prepriming complex is formed.2.Primase synthesizes RNA primer.DnaG is a primase.It binds the template and is activated by DnaB.The activated primase synthesizes RNA primer.3.DNA pol III elongates DNA and DNA pol I removes the prime

24、r.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.()2222 Two core enzymes()2 are held together by a complex.subunit:DNA synthesisDNA synthesis subunit:proofreading subunit:the sliding clamp A single h

25、oloenzyme is responsible for the synthesis of both leading strand and Okazaki fragments of lagging strand.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 str

26、and is looped out both leading and lagging strand synthesis move in the same direction.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 Okazak

27、i fragment.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.The large fragment is called klenow fragment.It has the polymerase activity and the 35 exonucl

28、ease activity.The 53 exonuclease removes the primer.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.4.Tus protein recognizes and binds to the TER site.That

29、 prevents the replication fork advancing.DNA replication terminates.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.GATC is a palindrome.The opposite strand

30、 also reads GATC in the 53 direction.5GATC3 3CTAG5Only at the full-methylated oriC can initiate replication.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

31、 methylated,about 10 minutes elapse before this can happen.6.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 negat

32、ive su-percoiling into DNA hence removing supercoiling.E 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.Section Four DNA Replication in Eukaryotes1.There are five types of com

33、mon eukaryotic DNA polymerases:,.2.Eukaryotic and prokaryotic enzymes and protein factors that participate in DNA replication at the replication fork are comparable.3.After the polymerase/primase complex initiates replication polymerase starts elongation.4.There are two mechanisms of removing primer

34、s.a.RNase HI and FEN1-dependent mechanism.b.helicase Dna2 and EFN1-dependent mechanism.5.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.6.Telomerase participates in

35、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.Thus genetic informati

36、on 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.The enzyme telomerase contains a short RNA

37、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.Section FiveDNA Rep

38、lication in Mitochondria and Phages1.mtDNA replicates in D-loops.2.Phages circular DNA replicates in rolling circle.Section Six The Repair of DNA Damage1.Physical or chemical agents may cause DNA damage.There are replication errors.There are several DNA repair systems in both prokaryotic and eukaryo

39、tic cells.2.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.The mismatched base is recogni

40、zed 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.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.3.DNA Damage R

41、epair 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.The photoreactivation repair system is a direct repair system.Direct repair can rep

42、air 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.b.Base Excision Repair SystemSingle base damages such

43、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).AP endonuclease hydrolyzes the phos-phordiester bond at the 5-end of the AP site.AP exonuclease cleaves the p

44、hosphor-diester bond at the 3-end of the AP site.and the deoxyribose-phosphate is re-moved.The gap left is filled with a nucleotide complementary to the template.DNA ligase makes the final phosphor-diester bond.c.Nucleotide Excision Repair SystemNucleotide excision repair system recog-nizes the dist

45、ortion of the DNA double helix.The distortion may be caused by TT,CT or CC dimer.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.UvrC makes nicks at bo

46、th 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.Xeroderma Pigmentosum(XP)Human NER system consists of a numberof XP proteins including XPA,XPB,XPC,XPD,XPF,XPG and E

47、RCC1.The complex of XP proteins scans the DNA,recognizes the lesion and removesa piece of damaged DNA about 25 nuc-leotides in length.The 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

48、of lacking of XP protein activities.That causes cancer and is known asxeroderma pigmentosum.d.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).Homologous recombina

49、tion(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:broken DNA5-33-5,5-33-5 intact homologous DNAThe broken region undergoes 5-exonuclease digestion.5-3 5-33-5 3-55-33-5 unwinding and strand inva

50、sion 5-3 5-3 3-5 -5 5-3 -33-5 DNA synthesis5-33-5-53-55-3 resolution of Holiday junctions5-33-53-55-3In 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