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1、Chapter 15DNA Biosynthesis and DNA Damage Repair,Section One The General Features of Replication of Chromosomal DNA,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-trat
2、ed 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 medi
3、um 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 D
4、NA 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 14N
5、H4Cl medium 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 semiconserv
6、ative man-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 stra
7、nds which 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
8、and comprise 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.,Summ
9、ary 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.,4.DNA replication is semidiscontinuous.The mechanism
10、 of DNA replication 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 temp
11、late 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
12、 joined by DNA ligase 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 factor
13、s.,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.,6.DNA replication needs priming.DNA polymerases cannot initiate DNA replication by starting a new DNA chain.They can only add nucleotides
14、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.,.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.Th
15、ere are also DNA priming 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 polymerase
16、s elongate DNA chains.DNA 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.mismat
17、ch repair.,Section Two Features of DNA Polymerases,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-close
18、d 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.,.The palm domain has three functions:a.polymerase activity b.to check the
19、 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 thumb domain keeps the primer-template junction in position in the active center and makes
20、the polymerase bind the substrates 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 R
21、eplication in E coli,1.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.,.DnaA protein forms a complex of 20-40 molecules,each bound to an ATP mole-c
22、ule,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.,.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 t
23、he single DNA strand.Gyrase introduces 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 elon
24、gates 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.()2222,Two core enzymes()2 are held together by a complex.subunit:DNA synthesis subunit:proofreading subunit:
25、the sliding clamp A single holoenzyme 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 th
26、e template of the lagging strand 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 elongate
27、s and completes another Okazaki 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 polym
28、erase activity and the 35 exonuclease 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 recogn
29、izes and binds to the TER site.That 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.,GAT
30、C is a palindrome.The opposite strand 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 daug
31、hter strand is also destined to become 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 ener
32、gy of ATP hydrolysis to introduce negative 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 i
33、n Eukaryotes,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.,3.After the polymerase/primase complex initiates replication polymerase starts elongation.4.Th
34、ere are two mechanisms of removing primers.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 activati
35、on of pre-RC.,6.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 o
36、f the lagging strand.,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
37、.,The 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 synth
38、esis leaving a 3-overhang.,Section FiveDNA Replication in Mitochondria and PhagesmtDNA replicates in D-loops.2.Phages circular DNA replicates in rolling circle.,Section Six The Repair of DNA Damage,1.Physical or chemical agents may cause DNA damage.There are replication errors.There are several DNA
39、repair systems in both prokaryotic and eukaryotic 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
40、 parental strand.,The 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.,Helicase UvrD and an exonuclease remove a piece of ssDNA that contains the error.DNA polymerase III fills the
41、 gap and DNA ligase seals the nick.,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.,The photoreactivation repair sys
42、tem 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.,b.
43、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).,AP endonuclease hydrolyzes the phos-phordiester bond at
44、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.,The gap left is filled with a nucleotide complementary to the template.DNA ligase makes the final phosphor-diester bond.,c.Nucleotide Excision Repair Syste
45、mNucleotide excision repair system recog-nizes the distortion 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.U
46、vrB recruits UvrC,an endonuclease.,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.,Xeroderma Pigmentosum(XP)Human NER system consists of a nu
47、mberof 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.,The gap is repaired by DNA polymeraseand DNA ligase.Patients having recessive mutations in XP protein genes
48、 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.,d.Recombinational repair(RR)system is responsible for repairing DNA double-strand breaks.The exchange of homologous regions between two DNA molecule
49、s is calledhomologous recombination(HR).,Homologous 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:,broken DNA5-33-5,5-33-5 intact homologous DNAThe broken region undergoes 5-exonuc
50、lease digestion.,5-3 5-33-5 3-55-33-5 unwinding and strand invasion,5-3 5-3 3-5-5 5-3-33-5 DNA synthesis,5-33-5-53-55-3 resolution of Holiday junctions,5-33-53-55-3,In E coli RecBCD protein complex hasboth nuclease and helicase activities.RecA protein participates in the formationof the Holliday jun
