Replication 13

2,110 views

Published on

Published in: Technology
1 Comment
0 Likes
Statistics
Notes
  • I LOVE SLIDESHARE WELCOME MORE
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Be the first to like this

No Downloads
Views
Total views
2,110
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
175
Comments
1
Likes
0
Embeds 0
No embeds

No notes for slide
  • 定點突變 是使用人工合成的引子,先與目標基因雜合 (2) ,而此引子的核 苷 酸序列與目標基因互補,但其上有一個核 苷 酸變異 (GTC→G C C) 。然後補滿雙股核酸 (3) ,再把此混成基因轉殖進入宿主,則宿主菌的複製系統將會因複製而產生兩種質體 (4) ,其一為原來的野生型,轉譯可得原來的蛋白質 (5) ;另一則為突變型,轉譯則得到突變的蛋白質,但只改變了一個指定位置的胺基酸 (6) 。
  • It is the phosphate group which gives DNA its acidic properties
  • 基因庫 的建構依基因來源的不同而有兩種方法,其一是上述由 mRNA 所得的 cDNA 來建庫 ( 上圖左 ) ,另一則由染色體 DNA 的限制 脢 片段來建庫 ( 上圖右 ) 。兩者在基因庫大小與其用途上,有相當的差別,要依使用目的如何而做適當選擇。 許多常用的生物或細胞,都有已經建立好的基因庫出售,只要買回來篩選,即可得到所要的基因。 當然,自己必須準備好適當的探針。 上圖雖然都使用質體當作載體 (vector) ,但是染色體基因庫中所攜帶的核酸片段都很大,因此多改用噬菌體基因,可以容納較長的片段。
  • 因為 限制脢可以辨認核酸序列上的特定序列,因此對某段固定的核酸,每次用相同限制脢所切的位置是固定的,所得到的大小片段也都是恆定的,這些片段可用電泳分離之,稱為限制脢圖譜。 當然,使用不同的限制脢會切在不同的地方,因此 A, B 兩種限制脢所切得的電泳圖譜也會不同。 若 同時 使用這兩種限制脢,則由所得到的圖譜,可推出這兩種限制脢在該段核酸上的切點位置。 例如上面的兩種限制脢分別切出 2 + 8 (A) 以及 3 + 7 (B) 兩種圖譜,合起來都是 10 kb ,對其切點的預測有兩種可能;但經由混合水解所得到的 2 + 3 + 5 片段,可以推出兩個限制脢切點之間相隔 5 kb ,若不做混合水解則無法得知。
  • 限制脢 所切出來的 sticky ends ,可以與另一相同的 sticky end 相連結,再用 DNA ligase 連結脢把各股接好,這使得 DNA 的剪接非常精準有效,是基因操作的最基本工具之一。任何兩個鈍端 (blunt ends) 也可相互連接,而且沒有專一性,但其連結效率非常低。 這些工具提供了在核酸序列上的精確剪接動作,可以像編輯錄音帶一樣,把所要的核酸片段剪出,然後接到另一段核酸上,開啟了基因操作的大門,科學家也快樂地打開了潘多拉的盒子。
  • Replication 13

    1. 1. 1DNA STRUCTUREDNA STRUCTUREANDANDREPLICATIONREPLICATIONDR.K.S.SODHIPROFESSORMMIMS&R
    2. 2. 2Central DogmaRNADNAProtein轉錄轉譯TranscriptionTranslationReplication複製逆轉錄ReverseTranscriptionJuang RH (2004) BCbasics
    3. 3. 33ARTHUR KORNBERG1958ARTHUR KORNBERG1958
    4. 4. 423 paternaldirectories23 maternalequivalentsTotal 35,000 filesReplicationNucleus23 x 2In 46 chromosomesHomologous chromosomesBefore celldivision3,000 MBCellNucleusJuang RH (2004) BCbasics
    5. 5. 5Site-directed mutagenesisCAGGTCCAGGCCCAGGCCCAG+ polymerase+ primerreplicationGCCCGGMutantThrtranslationWild typeGTCCAGValtranslationOnly one amino acid changedWild type proteinMutant proteinprimer(1)(2)(3)(5)(4)(6)Val → ThrSmith (1993)JuangRH(2004)BCbasics
    6. 6. 6AbbreviationsAbbreviations dsDNA.dsDNA. ssDNA.ssDNA. Ori.Ori. SSBsSSBs DnaK,dnaJ,dnaEDnaK,dnaJ,dnaE DNA Helicase.DNA Helicase. dRibosedRibose ApoptosisApoptosis Leading & Laging strands.Leading & Laging strands. Gyrase.Gyrase. Speed:100nts/sec. total 9 hours toSpeed:100nts/sec. total 9 hours tocomplete in a typical cell.complete in a typical cell. Double stranded DNADouble stranded DNA Single stranded DNASingle stranded DNA Origin of replication.Origin of replication. Single strand Binding.Single strand Binding. Heat shock proteins EHeat shock proteins E Unwind short segmenUnwind short segmen De-OxyriboseDe-Oxyribose Programmed cell deathProgrammed cell death One strech, multiple streches.One strech, multiple streches. Negative supercoiling using ATPNegative supercoiling using ATP
    7. 7. 7 cDNA asingle stranded DNA molecule thatcDNA asingle stranded DNA molecule thatis complementary to mRNA and isis complementary to mRNA and issynthesised from it by the action ofsynthesised from it by the action ofreverse transcriptase.reverse transcriptase. miRNAS micro RNAs 21-25 nucleotidemiRNAS micro RNAs 21-25 nucleotidelong.long. Sines Short interspread repeatSines Short interspread repeatsequences.sequences. Si RNA silencing RNA 21-25 nt length canSi RNA silencing RNA 21-25 nt length cancause gene knockdown.cause gene knockdown.
    8. 8. 8PROTEINS & FUNCTIONSPROTEINS & FUNCTIONS DNA polymerasesDNA polymerases Helicases.Helicases. Topoisomerases.Topoisomerases. DNA primaseDNA primase SSB proteinsSSB proteins DNA LigaseDNA Ligase Polymerisation.Polymerisation. Unwinding of DNA.Unwinding of DNA. Remove supercoiling.Remove supercoiling. Initiates synth. of RNAInitiates synth. of RNAprimer.primer. Prevent reanealing ofPrevent reanealing ofdsDNA.dsDNA. Seals the nick inSeals the nick inokazaki fragments.okazaki fragments.
    9. 9. 9REQUIREMENTSREQUIREMENTS Four activated precursors ofFour activated precursors ofdATP,dGTP,dCTP andTTP MgdATP,dGTP,dCTP andTTP Mg++++.. Template Strand.Template Strand. Primer with free3’-OH group(10-200)Primer with free3’-OH group(10-200) Elongation proceeds 5’—3’ direction.Elongation proceeds 5’—3’ direction. Removal of mismatched nucleotides.Removal of mismatched nucleotides. Error rate is less than 10Error rate is less than 10 -8-8 perbp. 3’3’hydroxyl group attack(nucleophillic) onpo4 of recently attached nucleotide.
    10. 10. 10DNADNA DNA stands for deoxyribose nucleic acid This chemical substance is present in thenucleus of all cells in all living organisms DNA controls all the chemical changeswhich take place in cells The kind of cell which is formed, (muscle,blood, nerve etc) is controlled by DNA The kind of organism which is produced(buttercup, giraffe, herring, human etc) iscontrolled by DNA The kind of organismwhich is produced (buttercup, giraffe,herring, human etc) is controlled by DNA
    11. 11. 11Ribose is a sugar, like glucose, but with only fivecarbon atoms in its moleculeDeoxyribose is almost the same but lacks oneoxygen atomBoth molecules may be represented by thesymbolRibose & deoxyriboseRibose & deoxyribose
    12. 12. 12The most common organic bases areAdenine (A)Thymine (T)Cytosine (C)Guanine (G)The basesThe bases
    13. 13. 13The deoxyribose, the phosphate and one of the basesadeninedeoxyribosePO4Combine to form a nucleotideNucleotidesNucleotides
    14. 14. 14A molecule ofDNA is formed bymillions ofnucleotides joinedtogether in a longchainPO4PO4PO4PO4sugar-phosphatebackbone + basesJoined nucleotidesJoined nucleotides
    15. 15. 15PO4PO4PO4PO4PO4PO4PO4PO4PO4PO4PO4PO4PO4PO4PO4PO42-stranded DNA2-stranded DNA
    16. 16. 16REPLICATION STEPSREPLICATION STEPS A. INITIATIONA. INITIATION B. ELONGATION.B. ELONGATION. C. TERMINATION.C. TERMINATION.
    17. 17. 17DNA ReplicationDNA Replication Priming:Priming:1.1. RNA primersRNA primers: before new DNA strands can: before new DNA strands canform, there must be small pre-existingform, there must be small pre-existingprimers (RNA)primers (RNA) present to start the addition ofpresent to start the addition ofnew nucleotidesnew nucleotides (DNA Polymerase)(DNA Polymerase)..2.2. PrimasePrimase: enzyme that polymerizes: enzyme that polymerizes(synthesizes) the(synthesizes) the RNA PrimerRNA Primer..
    18. 18. 18This DNA polymerase replaces the RNA primerwith DNA.This is a different type of DNA polymerasefrom the main DNA polymerase whichsynthesises DNA on a DNA template.Another DNA polymerase:
    19. 19. 19In E. coli the main enzyme is DNApolymerase III .And the enzyme that replaces the RNAprimer with DNA is DNA polymerase I.When the RNA primer has beenreplaced with DNA, there is a gapbetween the two Okazaki fragmentsand this is sealed by DNA ligase.
    20. 20. 20DNA ligase seals the gap left between Okazakifragments after the primer is removed. As theOkazaki fragments are joined, the new laggingstrand becomes longer and longer.DNA ligase:Location: At the replication fork.Function: Unwinds the DNA double helix.Helicase:
    21. 21. 21Location: On the template strands.Function: Synthesizes new DNA in the5 to 3 direction using the baseinformation on the template strand tospecify the nucleotide to insert on the newchain. Also does some proofreading; thatis, it checks that the new nucleotide beingadded to the chain carries the correct baseas specified by the template DNA.DNA polymerase:
    22. 22. 22The new DNA strand made discontinuouslyin the direction opposite to the direction inwhich the replication fork is moving.The new DNA strand made continuously inthe same direction as movement of thereplication fork.Lagging Strand:Leading strand:
    23. 23. 23If an incorrect base pair is formed,DNA polymerase can delete the newnucleotide and try again. In E. colithe enzyme used for all new DNAsynthesis except for the replacementof the RNA primers is DNApolymerase III. DNA polymerase Ireplaces the primers.
    24. 24. 24Location: On the template strand whichdictates new DNA synthesis away from thedirection of replication fork movement.Okazaki fragment:
    25. 25. 25Function: A building block for DNAsynthesis of the lagging strand. On onetemplate strand, DNA polymerasesynthesizes new DNA in a direction awayfrom the replication fork movement.Because of this, the new DNA synthesizedon that template is made in adiscontinuous fashion; each segment iscalled an Okazaki fragment.
    26. 26. 26Location: Wherever the synthesis of a newDNA fragment is to commence.Function: DNA polymerase cannot start thesynthesis of a new DNA chain, it can onlyextend a nucleotide chain primer. Primasesynthesizes a short RNA chain that is used asthe primer for DNA synthesis by DNApolymerase.Primase:
    27. 27. 27Location: On single-stranded DNA near thereplication fork. Function: Binds to single-stranded DNA to make it stable.Single-strand binding (SSB)proteins
    28. 28. 28Alternative models of DNA replication
    29. 29. 29DNA Replication 1 Models of DNA replication: -Meselson-StahlExperiment DNA synthesis and elongation DNA polymerases Origin and initiation of DNA replication Prokaryote/eukaryote models Telomere replication
    30. 30. 30Let us animate theLet us animate theprocess of replicationprocess of replication
    31. 31. 31
    32. 32. 32H bonds break Two strands seperate
    33. 33. 33Sugar phosphate backbone is made by joining the adjcentnucleotides ( DNA polymarase enzyme( ) )Nucleotides with Complementary bases are assembledalongside each strands
    34. 34. 34Two identical DNA molecules are formed
    35. 35. 351958: Matthew Meselson & Frank Stahl’s ExperimentSemiconservative model of DNA replication (Fig. 3.2)
    36. 36. 361955: Arthur KornbergWorked with E. coli.Discovered the mechanisms of DNA synthesis.Four components are required:1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)2. DNA template3. DNA polymerase (Kornberg enzyme)4. Mg 2+(optimizes DNA polymerase activity)1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)
    37. 37. 37Three main features of the DNA synthesisreaction:1. DNA polymerase I catalyzes formation of phosphodiester bondbetween 3’-OH of the deoxyribose (on the last nucleotide) andthe 5’-phosphate of the dNTP.• Energy for this reaction is derived from the release of two of thethree phosphates.2. DNA polymerase “finds” the correct complementary dNTP at eachstep in the lengthening process.• rate ≤ 800 dNTPs/second• low error rate3. Direction of synthesis is 5’ to 3’
    38. 38. 38DNA elongation
    39. 39. 39DNA elongation (Fig. 3.3a):
    40. 40. 40There are many different types of DNA polymerasePolymerasePolymerization(5’-3’)Exonuclease (3’-5’)Exonuclease(5’-3’)#CopiesI YES YES YES 400II YES NO YES?III YES YES YES20-40
    41. 41. 41 3’ to 5’ exonuclease activity = ability to removenucleotides from the 3’ end of the chain Important proofreading ability– Without proofreading error rate (mutation rate) is 1x 10-6– With proofreading error rate is 1 x 10-9 (1000-folddecrease) 5’ to 3’ exonuclease activity functions in DNAreplication & repair.
    42. 42. 42Eukaryotic enzymes: Five DNA polymerases from mammals. Polymerase α (alpha): nuclear, DNA replication,no proofreading Polymerase β (beta): nuclear, DNA repair, noproofreading Polymerase γ (gamma): mitochondria, DNArepl., proofreading Polymerase δ (delta): nuclear, DNA replication,proofreading
    43. 43. 43 Polymerase ε (epsilon): nuclear, DNArepair (?), proofreading Different polymerases for nucleus andmtDNA Some polymerases proofread; others do not. Some polymerases used for replication;others for repair
    44. 44. 44Origin of replication (e.g., the prokaryote example): Begins with double-helix denaturing into single-strands thus exposing thebases. Exposes a replication bubble from which replication proceeds in bothdirections.
    45. 45. 45Initiation of replication, major elements: Segments of single-stranded DNA are called templatestrands. Gyrase (a type of topoisomerase) relaxes the supercoiledDNA. Initiator proteins and DNA helicase binds to the DNA atthe replication fork and untwist the DNA using energyderived from ATP (adenosine triphosphate).(Hydrolysis of ATP causes a shape change in DNAhelicase) DNA primase next binds to helicase producing acomplex called a primosome (primase is required forsynthesis),
    46. 46. 46 Primase synthesizes a short RNA primer of 10-12nucleotides, to which DNA polymerase III addsnucleotides. Polymerase III adds nucleotides 5’ to 3’ on both strandsbeginning at the RNA primer. The RNA primer is removed and replaced with DNA bypolymerase I, and the gap is sealed with DNA ligase. Single-stranded DNA-binding (SSB) proteins (>200)stabilize the single-stranded template DNA during theprocess.
    47. 47. 47
    48. 48. 48DNA replication is continuous on the leading strand and semidiscontinuous on thelagging strand:Unwinding of any single DNA replication fork proceeds in one direction.The two DNA strands are of opposite polarity, and DNA polymerases onlysynthesize DNA 5’ to 3’.Solution: DNA is made in opposite directions on each template.•Leading strand synthesized 5’ to 3’ in the direction ofthe replication fork movement.continuousrequires a single RNA primer•Lagging strand synthesized 5’ to 3’ in the oppositedirection.semidiscontinuous (i.e., not continuous)requires many RNA primers
    49. 49. 493Polymerase III5’ →3’Leading strandbase pairs5’5’3’3’Supercoiled DNA relaxed by gyrase & unwound by helicase + prHelicase+Initiator ProteinsATPSSB ProteinsRNA Primerprimase2Polymerase IIILagging strandOkazaki Fragments1RNA primer replaced by polymerase I& gap is sealed by ligase
    50. 50. 50
    51. 51. 51
    52. 52. 52Two Libraries : cDNA Library vs Genomic LibrarymRNAcDNAReverse transcriptionChromosomal DNARestriction digestionGenes in expression Total GeneCompletegene Gene fragmentsSmallerLibraryLargerLibraryVector:Plasmid or phageVector: PlasmidJuang RH (2004) BCbasics
    53. 53. 53Restriction Mapping of DNAA B 10 kb8 kb2 kbA7 kb3 kbB5 kb3 kb2 kbA+BCK A B A+B MRestrictionenzymesJuang RH (2004) BCbasics
    54. 54. 54The Specific Cutting and Ligation of DNAGAATTCCTTAAGGAATTCCTTAAGGCTTAAAATTCGAATTCGGCTTAAGCTTAAAATTCGGCTTAAAATTCGGCTTAAAATTCGEcoRIDNA LigaseEcoRI sticky end EcoRI sticky endJuang RH (2004) BCbasics
    55. 55. 55
    56. 56. 56DNA ligase seals the gaps between Okazaki fragments with aphosphodiester bond (Fig. 3.7)TIME: E.coli 30 minutes,Humans: 24 Hours.
    57. 57. 57Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.Fig. 3.5 - Model of DNA replication
    58. 58. 58Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.Fig. 3.5 - Model of DNA replication
    59. 59. 59Concepts and terms to understand:Why are gyrase and helicase required?The difference between a template and a primer?The difference between primase and polymerase?What is a replication fork and how many are there?Why are single-stranded binding (SSB) proteins required?How does synthesis differ on leading strand and lagging strand?Which is continuous and semi-discontinuous?What are Okazaki fragments?
    60. 60. 60Replication ofcircular DNA inE. coli (3.10):1. Two replication forks result ina theta-like (θ) structure.2. As strands separate, positivesupercoils form elsewhere inthe molecule.3. Topoisomerases relievetensions in the supercoils,allowing the DNA to continueto separate.
    61. 61. 61Rolling circle model of DNAreplication (3.11):1. Common in severalbacteriophages includingλ.2. Begins with a nick at theorigin of replication.3. 5’ end of the molecule isdisplaced and acts asprimer for DNA synthesis.4. Can result in a DNAmolecule many multiplesof the genome length (andmake multiple copiesquickly).5. During viral assembly theDNA is cut into individualviral chromosomes.
    62. 62. 62DNA replication in eukaryotes:Copying each eukaryotic chromosome during the S phase of the cell cyclepresents some challenges:Major checkpoints in the system1. Cells must be large enough, and the environment favorable.2. Cell will not enter the mitotic phase unless all the DNA has replicated.3. Chromosomes also must be attached to the mitotic spindle for mitosisto complete.4. Checkpoints in the system include proteins call cyclins and enzymescalled cyclin-dependent kinases (Cdks).
    63. 63. 63• Each eukaryotic chromosome is one linear DNAdouble helix• Average ~108base pairs long• With a replication rate of 2 kb/minute, replicatingone human chromosome would require ~35 days.• Solution ---> DNA replication initiates at manydifferent sites simultaneously.Fig. 3.14
    64. 64. 64Fig. 3.13 - Replication forks visible in Drosophila
    65. 65. 65(or telomeres What about the ends ) of linearchromosomes?DNA polymerase/ligase cannot fill gap at end of chromosome afterRNA primer is removed. this gap is not filled, chromosomeswould become shorter each round of replication!Solution:1. Eukaryotes have tandemly repeated sequences at the ends oftheir chromosomes.2. Telomerase (composed of protein and RNA complementary tothe telomere repeat) binds to the terminal telomere repeat andcatalyzes the addition of of new repeats.3. Compensates by lengthening the chromosome.4. Absence or mutation of telomerase activity results inchromosome shortening and limited cell division.
    66. 66. 66Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.Fig. 3.16 Synthesis of telomeric DNA by telomerase
    67. 67. 67Final Step - Assembly into Nucleosomes:• As DNA unwinds, nucleosomes must disassemble.• Histones and the associated chromatin proteins mustbe duplicated by new protein synthesis.• Newly replicated DNA is assembled into nucleosomesalmost immediately.• Histone chaperone proteins control the assembly.Fig. 3.17
    68. 68. 6868DNA DAMAGEDNA DAMAGEAND REPAIRAND REPAIR
    69. 69. 69DNA DAMAGE1. SPONTANEOUS2. ENVIRONMENTALAGENTS3. REPLICATIONPHYSICALCHEMICALBIOLOGICAL
    70. 70. 70Mis-incorporationofbasesChemicals UV-radiationX-radiationSpontaneousDe-aminationof basesCAUSESOFDAMAGE
    71. 71. 71SingleBasealterationsCrossLinkageChainbreaksTwoBasealterationsTYPESOFDAMAGE
    72. 72. 721.1. SINGLE BASE ALTERATIONSSINGLE BASE ALTERATIONS DEPURINATIONDEPURINATION DEAMINATION OF CYTOSINE TO URACILDEAMINATION OF CYTOSINE TO URACIL DEAMINATION OF ADENINE TODEAMINATION OF ADENINE TOHYPOXANTHINEHYPOXANTHINE ALKYLATION OF BASESALKYLATION OF BASES INSERTION OR DELETION OF NUCLEOTIDESINSERTION OR DELETION OF NUCLEOTIDES BASE ANALOG INCORPORATIONBASE ANALOG INCORPORATION
    73. 73. 732.2. TWO BASE ALTERATIONSTWO BASE ALTERATIONS UV INDUCED THYMINE-THYMINEUV INDUCED THYMINE-THYMINEDIMERSDIMERS BIFUNCTIONAL ALKYLATING AGENTSBIFUNCTIONAL ALKYLATING AGENTS
    74. 74. 743.3. CHAIN BREAKSCHAIN BREAKS IONIZING RADIATION INDUCEDIONIZING RADIATION INDUCED RADIOACTIVE DISINTEGRATION OFRADIOACTIVE DISINTEGRATION OFBACKBONE ELEMENTBACKBONE ELEMENT FREE-RADICAL INDUCEDFREE-RADICAL INDUCED
    75. 75. 754. CROSS LINKAGE4. CROSS LINKAGE BETWEEN BASES IN SAME ANDBETWEEN BASES IN SAME ANDOPPOSITE STRANDOPPOSITE STRAND BETWEEN DNA AND PROTEINBETWEEN DNA AND PROTEINMOLECULESMOLECULES
    76. 76. 761. Proofreadingand editing2. MismatchRepairsystem3. BaseExcisionrepair4. NucleotideExcisionRepair5. Photo-ReactivationOrDirectrepair6. DoubleStrandBreakRepair7.Transcription-Coupledrepair
    77. 77. 771. PROOF READING AND1. PROOF READING ANDEDITINGEDITING Despite doubleDespite doublemonitoring duringmonitoring duringreplication, first at time ofreplication, first at time ofincorporation of bases andincorporation of bases andsecond by later follow upsecond by later follow upenergy requiringenergy requiringprocesses, some mispairedprocesses, some mispairedbases persist which havebases persist which haveto be removed by otherto be removed by otherenzyme systems.enzyme systems. The proof reading activityThe proof reading activityis carried out by 3’-5’is carried out by 3’-5’exonuclease activities ofexonuclease activities ofDNA polymerase III andDNA polymerase III andI.I.
    78. 78. 782. MISMATCH REPAIR SYSTEM2. MISMATCH REPAIR SYSTEM This mechanism operates immediately afterThis mechanism operates immediately afterDNA replication.DNA replication. Sometimes the replication errors escape theSometimes the replication errors escape theDNA proofreading function. This mechanismDNA proofreading function. This mechanismchecks for the correction of escaped bases.checks for the correction of escaped bases. Specific proteins scan the newly synthesizedSpecific proteins scan the newly synthesizedDNA by the following mechanisms:DNA by the following mechanisms:1.1. Identification of mismatched strandIdentification of mismatched strand2.2. Repair of mispaired base.Repair of mispaired base.
    79. 79. 79 In error detection,In error detection,parent strand isparent strand isidentified first with theidentified first with thehelp of GATC-help of GATC-sequences, that occursequences, that occurapprox. once after everyapprox. once after everythousand nucleotides.thousand nucleotides. It is methylated atIt is methylated atadenine residue.adenine residue. The methylation doesThe methylation doesnot occur immediatelynot occur immediatelyafter replication. So, theafter replication. So, thenew strand is notnew strand is notmethylated and is easilymethylated and is easilyidentified.identified.
    80. 80. 80 Secondly, on the new strand, GATC-endonucleaseSecondly, on the new strand, GATC-endonuclease‘nicks’ the mismatched strand.‘nicks’ the mismatched strand. This faulty strand is digested by exonuclease.This faulty strand is digested by exonuclease. An extensive region, from the mismatched area tillAn extensive region, from the mismatched area tillthe next GATC-sequence is removed.the next GATC-sequence is removed. This gap is filled by the DNA polymerase I, in 5’-3’This gap is filled by the DNA polymerase I, in 5’-3’direction.direction. Clinical significance-Clinical significance- A defect in mismatch repairA defect in mismatch repairin humans has been known to cause hereditary non-in humans has been known to cause hereditary non-polyposis colon cancer (HNPCC).polyposis colon cancer (HNPCC).
    81. 81. 813. BASE EXCISION REPAIR3. BASE EXCISION REPAIR This mechanism operates all the time in the cells.This mechanism operates all the time in the cells. The bases of DNA can be altered:The bases of DNA can be altered:a)a) SpontaneouslySpontaneously -- cytosine uracil.cytosine uracil.b)b) Deaminating compounds -Deaminating compounds - like NO, which is formedlike NO, which is formedfrom nitrosamines,nitrites, and nitrates.from nitrosamines,nitrites, and nitrates.- NO is a potent de-aminating compound, that converts:- NO is a potent de-aminating compound, that converts:i.i. Ctytosine uracilCtytosine uracilii.ii. Adenine hypoxanthineAdenine hypoxanthineiii.iii. Guanine xanthineGuanine xanthinec)c) Bases can also be lost spontaneously -Bases can also be lost spontaneously - approximately,approximately,10,000 purine bases are lost spontaneously per cell per10,000 purine bases are lost spontaneously per cell perday.day.
    82. 82. 82 Following mechanismsFollowing mechanismsoperate to correct such baseoperate to correct such basealterations or losses:alterations or losses:1.1. Removal of abnormalRemoval of abnormalbases-bases- Abnormal bases areAbnormal bases arerecognized by specificrecognized by specificglycosylasesglycosylases..-they hydrolytically cleave-they hydrolytically cleavethem from deoxy-ribose-them from deoxy-ribose-phosphate backbone of thephosphate backbone of thestrand.strand.-this results in either A-this results in either Apurinicpurinicor Apyrimidinicor Apyrimidinic site,site,referred to asreferred to as AP- siteAP- site..
    83. 83. 832.2. Repair of AP-site -Repair of AP-site - AP-endonucleaseAP-endonuclease recognizes therecognizes theempty site and starts excision by making a cut at 5’-end ofempty site and starts excision by making a cut at 5’-end ofAP-site.AP-site.-- deoxy-ribose-phosphate lyasedeoxy-ribose-phosphate lyase removes the single, empty,removes the single, empty,sugar-phosphate residue and gap is finally filled bysugar-phosphate residue and gap is finally filled by DNADNApolymerase Ipolymerase I and nick is sealed byand nick is sealed by DNA ligaseDNA ligase..NOTE..NOTE.. By the similar series of steps involving initially theBy the similar series of steps involving initially therecognition of the defect, the alkylated bases and baserecognition of the defect, the alkylated bases and baseanalogs can be removed from DNA. And thus, DNAanalogs can be removed from DNA. And thus, DNAreturns to its original information content.returns to its original information content. This mechanism is efficient only for replacement of aThis mechanism is efficient only for replacement of asingle base but is not efficient for replacing regions ofsingle base but is not efficient for replacing regions ofdamaged DNA.damaged DNA.
    84. 84. 84Recognition and excision ofRecognition and excision ofdefectdefect(eg:-UV induced dimers)(eg:-UV induced dimers) First,a UV-specificFirst,a UV-specificendonuclease recognizes theendonuclease recognizes thedimer and cleaves thedimer and cleaves thedamaged strand atdamaged strand atphosphodiester bonds on bothphosphodiester bonds on both5’ side and 3’ side of the5’ side and 3’ side of thedimer.dimer. The damaged oligonucleotideThe damaged oligonucleotideis released, leaving a gap inis released, leaving a gap inthe DNA strand that formerlythe DNA strand that formerlycontained the dimer.contained the dimer. This gap is filled byThis gap is filled by DNADNApolymerase Ipolymerase I and nick isand nick issealed bysealed by DNA ligaseDNA ligase..
    85. 85. 855. PHOTO-REACTIVATION OR5. PHOTO-REACTIVATION ORDIRECT REPAIRDIRECT REPAIR This is also called ‘This is also called ‘lightlightinduced repair’.induced repair’. The enzyme photo-The enzyme photo-reactivating enzyme (PRreactivating enzyme (PRenzyme) brings about anenzyme) brings about anenzymatic cleavage ofenzymatic cleavage ofthymine dimers activated bythymine dimers activated bythe visible lightthe visible light It leads to restoration ofIt leads to restoration ofmonomeric condition.monomeric condition. Co-enzymes required for theCo-enzymes required for thereaction are FADH2 andreaction are FADH2 andTHF.THF.
    86. 86. 866. DOUBLE STRAND BREAK6. DOUBLE STRAND BREAKREPAIRREPAIR High energy radiation, oxidative free radicals or someHigh energy radiation, oxidative free radicals or somechemotherapeutic agents bring about double-strandedchemotherapeutic agents bring about double-strandedbreaks in DNA, or may also occur naturally duringbreaks in DNA, or may also occur naturally duringnaturally during gene rearrangements.naturally during gene rearrangements. They are potentially lethal to the cell.They are potentially lethal to the cell. They cannot be repaired by excising single strand andThey cannot be repaired by excising single strand andusing the other strand as template to replace missingusing the other strand as template to replace missingnucleotides.nucleotides. It is repaired by 2 ways:It is repaired by 2 ways:1.1. Non-homologous end-joining repair.Non-homologous end-joining repair.2.2. Homologous recombination repair.Homologous recombination repair.
    87. 87. 871.1. Non-homologous end joining repair-Non-homologous end joining repair- In this system the two ends of DNA areIn this system the two ends of DNA arebrought together by a group of proteins andbrought together by a group of proteins andthereby the ends are re-ligated.thereby the ends are re-ligated. This system does not require that the 2 DNAThis system does not require that the 2 DNAsequences have any homology.sequences have any homology.2.2. Homologous recombination repair-Homologous recombination repair- This system uses the enzymes that normallyThis system uses the enzymes that normallyperform genetic recombination betweenperform genetic recombination betweenhomologous chromosomes during meiosis.homologous chromosomes during meiosis. This is called sister-strand exchange.This is called sister-strand exchange.
    88. 88. 88
    89. 89. 897. TRANSCRIPTION COUPLED7. TRANSCRIPTION COUPLEDREPAIRREPAIRWhen RNA polymerase transcribes aWhen RNA polymerase transcribes agene, as it encounters a damagedgene, as it encounters a damagedregion, the transcription stops.region, the transcription stops.The excision repair enzymes repairThe excision repair enzymes repairthe area and then transcriptionthe area and then transcriptionresumes.resumes.
    90. 90. 90CLINICALCLINICALDISORDERSDISORDERS
    91. 91. 911.1. XERODERMAXERODERMAPIGMENTOSAPIGMENTOSA Autosomal recessive in nature.Autosomal recessive in nature. UV- specific exonuclease is deficient.UV- specific exonuclease is deficient. Cutaneous hypersensitivity to UV-rays.Cutaneous hypersensitivity to UV-rays. Blisters on skin.Blisters on skin. Hyperpigmentation.Hyperpigmentation. Corneal ulcer.Corneal ulcer. Death occurs due to formation of cancers ofDeath occurs due to formation of cancers ofskin.skin.
    92. 92. 922. ATAXIA TELANGIECTASIA2. ATAXIA TELANGIECTASIA Autosomal recessive inAutosomal recessive innature.nature. Increased sensitivity to X-Increased sensitivity to X-rays and UV-rays.rays and UV-rays. Progressive cerebellarProgressive cerebellarataxia.ataxia. Oculo-cutaneousOculo-cutaneoustelangiectasia.telangiectasia. Frequent sino-pulmonaryFrequent sino-pulmonaryinfections.infections. Lympho-reticular neoplasm.Lympho-reticular neoplasm.
    93. 93. 933. BLOOM’S SYNDROME3. BLOOM’S SYNDROME Chromosomal breaks orChromosomal breaks orrearrangements are seen.rearrangements are seen. Defect lies in DNA helicase orDefect lies in DNA helicase orligase.ligase. Facial erythmia.Facial erythmia. Photosensitivity.Photosensitivity. Lympho-reticularLympho-reticularmalignancies.malignancies.
    94. 94. 944.FANCONI SYNDROME4.FANCONI SYNDROME Lethal aplastic anaemia,due to defectiveLethal aplastic anaemia,due to defectiveDNA repair.DNA repair. Cells can not repair interstrand cross-links,Cells can not repair interstrand cross-links,or damage induced by X-Rays.or damage induced by X-Rays.
    95. 95. 955.Cockayne’sSyndrome5.Cockayne’sSyndrome&Retinoblastoma&Retinoblastoma Defects in DNA repair.Defects in DNA repair. Cells from patients with someCells from patients with somechromosomal abnormalities eg Downchromosomal abnormalities eg DownSyndrome may also show aberrant DNASyndrome may also show aberrant DNArepair.repair.
    96. 96. 96INHIBITOR OF DNAINHIBITOR OF DNAREPLICATIONREPLICATION Anthracyclines cause chainAnthracyclines cause chainbreakageSu.bstances that act directly onbreakageSu.bstances that act directly onDNA Polymerases eg. Acyclovir inhibitsDNA Polymerases eg. Acyclovir inhibitsthe DNA polymerase of herpes simplex.the DNA polymerase of herpes simplex. 2’-dideoxyazidocytidine is a inhibitor of2’-dideoxyazidocytidine is a inhibitor ofbacterial primase,andbacterial primase,andcournermycin,novobiocin,oxolinic acid andcournermycin,novobiocin,oxolinic acid andnalidixic acid are effective inhibitor of DNAnalidixic acid are effective inhibitor of DNAgyrase in bacteria.gyrase in bacteria.
    97. 97. 97TOPOISOMERASE ITOPOISOMERASE IINHIBITORINHIBITOR Topoisomerase is essential for DNA replicationTopoisomerase is essential for DNA replicationand cell growth.and cell growth. Certain drugs produces double strand breaks inCertain drugs produces double strand breaks inDNA that are irreversible and can lead to cellDNA that are irreversible and can lead to celldeath.death. Eg. Quilnolne antibiotics,anthracyclines activeEg. Quilnolne antibiotics,anthracyclines activefor treatment of lung, ovarian and colorectalfor treatment of lung, ovarian and colorectalcancer.The Camptothecins were discoveredcancer.The Camptothecins were discoveredfrom extract of tree Camptotheca acuminata.from extract of tree Camptotheca acuminata.
    98. 98. 98THE ENDkingkul@yahoo.co.in

    ×