Dna replication part i

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Dna replication part i

  1. 1. DNA REPLICATION PART-IByDr. Ichha PuraKUniversity ProfessorDepartment of BotanyRanchi Women’s College,Ranchiwww.dripurak.com6/15/2013DNA REPLICATION Part-I1
  2. 2. DNA REPLICATIONINTRODUCTIONIMPORTANCE OF REPLICATIONTHE REPLICATION FACTORY•MODE OF REPLICATION : SEMI CONSERVATIVE•EXPERIMENTAL EVIDENCE•PHASE OF REPLICATION•PLACE OF REPLICATION•DIRECTION OF REPLICATION•INITIATION OF REPLICATION : PROKARYOTES•EUKARYOTIC INITIATION OF REPLICATION•PROTEINS AND ENZYMES INVOLVED IN REPLICATION•REPLICATION FORK6/15/2013DNA REPLICATION Part-I2
  3. 3. DNA replication is a fundamental process by which the parent DNAduplex copies itself and become duplicated and produce two daughterDNA duplexes .Both strands of DNA ladder separate and act as template, onto whichnew daughter strands are assembled, for which complementarydeoxyribonucleotides are taken from pool of deoxyribonucleotides.The two DNA duplexes that emerge from this process receive only oneside of original DNA and other side newly synthesized.6/15/2013DNA REPLICATION Part-I3
  4. 4. IMPORTANCE OF REPLICATIONIt is one of the most vital processes. It provides means by whichgenetic instructions can be transmitted from one (parent ) cell to itstwo daughter cells or from one individual to its offsprings becauseduring Replication Parent DNA duplex is able to make its twoidentical copies or Replica and It is now well known that DNA is theGenetic Material,able to transmit information over generations.6/15/2013DNA REPLICATION Part-I4
  5. 5. 6/15/2013DNA REPLICATION Part-I5DNA TemplateNew DNAPARENTAL DNASemi-conservativeModel:Watson and Crickpredicted : the twostrands of theparental moleculeseparate, and eachfunctions as atemplate for synthesisof a newcomplementarystrand.
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  8. 8. THE REPLICATION FACTORYReplication proteins are clustered together in particular locations in thecell and may therefore be regarded as Replication Factory thatmanufactures DNA copies.The DNA to be copied is fed through the factory, much as a reel of film isfed through a movie projector. The incoming DNA double helix is splitinto two single strands and each original strand becomes half of a newdouble helix. Because each resulting DNA double helix retains onestrand of the original DNA. DNA replication is said to be semi-conservative6/15/2013DNA REPLICATION Part-I8
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  10. 10. MODE OF REPLICATION :Three possibilities have been suggested as mode for replication :•Conservative Of the two duplexes produced after replication one isentirely parental and other entirely new•Semi-conservative Both the two duplexes formed after replicationcontain one parental strand and one newly synthesized strand. ParentDNA double helix is not conserved as entity, But one parental strand isconserved in both daughter double helices in each generation and itcontinues for many generations.•Dispersive Both strands of parental molecule break at random, afterreplication the parental and daughter pieces are joined randomly.6/15/2013DNA REPLICATION Part-I10
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  12. 12. Of the various ideas proposed regarding mode of replication viz.conservative, semi-conservative and dispersive, the semi-conservative mode is highly accepted , which indicates that onehalf of the parent duplex is transmitted to each daughter duplex. Ithas been theoretically predicted by Watson and Crick ( 1953) onthe basis of double helical nature and complementary sequence ofbases on two strands of DNA.6/15/2013DNA REPLICATION Part-I12Semi conservative mode of replication has been experimentallyproved by Maselson and Stahl (1958) using 15N in the nutrientmedium for culturing E.coli and monitoring the density of DNA aftereach replication
  13. 13. EXPERIMENTAL EVIDENCESteps in the Experiment1.E. coli cells were grown in culture medium having NH4Cl (with15N)as Nitrogen source for 14 generations, so that all DNA basesbecome labeled. This heavier ( High density) DNA settlesdifferently after centrifugation than the lighter (Low Density ) DNAhaving 14 N.2. E.coli cells were then removed ,washed and transferred toculture medium having NH4Cl ( with 14 N) as Nitrogen source.3. Some cells of E. coli were removed after every 30 minutes andDNA was extracted and examined for density by centrifugationwith Cesium Chloride solution followed by sedimentation. ( E colitakes 30 minutes for one division)6/15/2013DNA REPLICATION Part-I13
  14. 14. The results were observed :4. After first generation only one band was observed by ultravioletabsorption(260 nm) and it showed density intermediate betweenheavier ( DNA 15 N) and lighter (DNA 14 N ) DNA s.5. After two generations two bands were seen, one comparable toIntermediate (Hybrid) DNA density and other of Normal ( DNA 14 N )density.6. In all subsequent generations two bands appeared, but intensity ofHybrid density band gradually decreased and band of lighter DNAgradually increased as expected by Semiconservative mode ofreplication.6/15/2013DNA REPLICATION Part-I14
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  18. 18. 6/15/2013DNA REPLICATION Part-I1815 N 14 N15 N+14 NHybrid
  19. 19. 6/15/2013DNA REPLICATION Part-I1915 NHybrid14 N
  20. 20. PHASE OF REPLICATIONTakes place during ‘S’ (Synthetic) phase of the preparatory stage ofcell cycle which commences after G 1 and is followed by G 2. Thepreparatory stage is also known as Resting stage as cell is supposed tobe not active in division. It is also known as Interphase as it is theperiod in between two cell divisions. During this period cell ismetabolically active.6/15/2013DNA REPLICATION Part-I20
  21. 21. Eukaryotic Replication having many originsPLACE OF REPLICATIONIt starts at origins of replication (Ori), appears as bubble underelectron microscope, ultimately extend in the form of ‘Y’shaped replication fork.6/15/2013DNA REPLICATION Part-I21
  22. 22. Bubble : 2 Replication Forks6/15/2013DNA REPLICATION Part-I22
  23. 23. CIRCULAR BACTERIALCHROMOSOME UNDERGOINGBIDIRECTIONALSEMICONSERVATIVEREPLICATION6/15/2013DNA REPLICATION Part-I23
  24. 24. 6/15/2013DNA REPLICATION Part-I24PROKARYOTIC DNA REPLICATIONParent duplex Daughter duplexes
  25. 25. 6/15/2013DNA REPLICATION Part-I25DIRECTION OF REPLICATIONReplication takes place in bi-directional way, proceeds on bothsides of origin of replication ( Ori), both in pro as well aseukaryotes. DNA synthesis takes place in 5’--------> 3’ direction andthe template is read in 3’ --------> 5’ direction so two newlysynthesized stretches of nucleotide chains must grow in oppositedirection . On one direction new strand grows towards fork and onother strand away from the fork.
  26. 26. 6/15/2013DNA REPLICATION Part-I26Thetalike configurationassumed byreplicating,circular,no-enddouble helical DNAmolecule of Escherichiacoli. Arrows indicate the tworeplication forks. Thesehave progressedbidirectionally from a singlereplication bubbleIn Bacteria, as DNA is circular and there is single (Ori), bi-directionalreplication gives a shape.
  27. 27. Pattern of replicationof long DNAmolecule with morethan one origin ofreplication.Replication proceedsout bidirectionallyfrom each origin6/15/2013DNA REPLICATION Part-I27
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  29. 29. Origin of replication in E.coliReplication initiates at a Unique site onthe E.coli chromosome ,designated asori.First event is the binding of an initiatorprotein to ori DNA , which leads topartial unwinding of the DNA doublehelix giving two templatesDNA continues to unwind by theaction of Helicase and singlestranded DNA binding proteins andRNA primers synthesized by PrimaseThe two replication forks formed atthe origin then move in oppositedirections along the circular DNAmolecule6/15/2013DNA REPLICATION Part-I29
  30. 30. INITIATION OF REPLICATION : FACTORS INVOLVEDProkaryotes : Eubacterial cell : E.coliInitiation begins at A -T rich sequences at Ori C in E.coli. The maincomponents of the replication complex formation are ORC(Origin Recognition Complex), factors Dna A, Dna B, Dna C, HUand enzyme helicase. Dna A binding site consists of 9 bp repeats of5’TGTGAATAA 3’, it binds to the Ori C , sets the platform and promotesdouble helical opening, it also loads Dna B .6/15/2013DNA REPLICATION Part-I30
  31. 31. Binding of Dna A protein appears to be the key event ,it appears asellipsoidal mass (Dna A-Ori C ) under electron microscope. Dna Bhas 5’ 3’ helicase activity and is also activator of primase Dna Ccomplex. HU is another double stranded binding protein.As ORC binds DNA with the help of different protein factors, thedouble helical structure opens in the form of small bubble by theenzyme helicase. Once double helical structure is opened, it isstabilized by single stranded DNA binding proteins.6/15/2013DNA REPLICATION Part-I31
  32. 32. EUKARYOTIC INITIATION OF REPLICATION : FACTORSINVOLVEDEukaryotic DNA is complexed with proteins, is assembled aschromosomes. Eukaryotic DNA is many times larger thanprokaryotic DNA and therefore have multiple origin ofreplication (ori) sites. The average human chromosome contains150x106 nucleotide pairs, which are copied at about 50 bp persec speed. But due to presence of multiple origin of replication,whole genome is replicated in 1 hour. In order to initiatereplication at these multiple sites a pre replicative complex isformed having the following components :6/15/2013DNA REPLICATION Part-I32
  33. 33. ORC ( Origin Recognition Complex ) ,these remain bound toDNA through out the Initiation process and is a six subunitcomplex. It has affinity for single stranded DNA. Other factorshelp ORC in identifying the ori sites. Its binding is coupled withATP hydrolysis.CdC6 protein associates with ORC and help MCM proteins toassociate with chromatin.Cdt 1 protein is identified as key factor in Pre-Rc assembly, somutations in Cdt 1 (In vitro studies ) results in a block to DNAreplication.6/15/2013DNA REPLICATION Part-I33
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  36. 36. MCM (Mini chromosome Maintenance ) proteins - DNA is coatedwith these proteins with the help of CdC6 and Cdt1. Oncereplication begins in S phase,Cdt1 and CdC6 leave the ORCs andMCM proteins remain in front of Replication fork on both sidesand all these help in stabilizing replication fork and its extensionhaving single stranded DNA.DNA synthesis begins with the activity of DNA helicase whichcauses melting of hydrogen bonds between base pairs ( A- T andG-C) . As a result two single stranded structures with theirexposed nucleotides are produced.6/15/2013DNA REPLICATION Part-I36
  37. 37. These single strands have a tendency to wound again (makebase pairs) and to avoid this single stranded DNA bindingproteins play important role. They also protect the singlestranded structures from nucleases. After this DNA polymerasecomes into action. It selects the complementary nucleotidefrom a mixture of dNTPs ( dATP,dCTP, dGTP,dTTP) and add tothe template strand and also establish phosphodi-ester bondsbetween successive nucleotides.6/15/2013DNA REPLICATION Part-I37
  38. 38. PROTEINS AND ENZYMES INVOLVED INREPLICATIONMany enzymes and proteins are involved in the process of DNAReplication to unwind double helix,replication fork stabilizationand synthesizing new DNA strand reading the template.viz.Helicase, SSB Protein, Primase, The sliding Clamp, DNAPolymerase, Rnase H and DNA Ligase. Each enzymes has aspecific role.DNA replication requires a variety of proteins. Each proteinperforms a specific function in the production of the new DNAstrands.Helicase, made of six proteins arranged in a ring shape, unwindsthe DNA double helix into two individual strands.6/15/2013DNA REPLICATION Part-I38
  39. 39. Single-strand binding proteins, or SSBs, are tetramers that coat thesingle-stranded DNA. This prevents the DNA strands fromreannealing to form double-stranded DNA. Primase is an RNApolymerase that synthesizes the short RNA primers needed to startthe strand replication process. DNA polymerase is a hand-shapedenzyme that strings nucleotides together to form a DNA strand. Thesliding clamp is an accessory protein that helps hold the DNApolymerase onto the DNA strand during replication. RNAse Hremoves the RNA primers that previously began the DNA strandsynthesis. DNA ligase links short stretches of DNA together tocreate one long continuous DNA strand.6/15/2013DNA REPLICATION Part-I39
  40. 40. 6/15/2013DNA REPLICATION Part-I40Enzymes and Proteins Involved in DNAReplication
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  42. 42. DNA Gyrase ( Type II Topoisomerase). Helps unwinding by DNAhelicase. Reduces the tension caused by super coil formation duringunwinding. For it single nick is created by breaking the phosphodiesterbond on one of the strand, which helps to release tension .Topoisomerases have both nuclease (strand cutting) and ligase (strandresealing) activities.DNA helicase Helps in dissolving ‘H’ bonds between base pairs andresult in separating the two strands near origin of replication, whichgradually extends. Helicase as it requires energy to open the duplex isassociated with hydrolysis of ATP.Singe stranded DNA binding Proteins Maintain the stability ofreplication fork by binding the separated strands on both sides andkeeping them apart to avoid rewinding.These proteins also protect theexposed nucleotides on the separated strands against nucleases.6/15/2013DNA REPLICATION Part-I42
  43. 43. DNA Polymerase Proceeds along the single strandedtemplates , recruit complementary dNTPs, form Hydrogenbonds with their appropriate complementary base present onthe template and catalyze phosphodiester bond with previousnucleotide of the same strand.In prokaryotes DP III isresponsible for synthesis of new DNA strands .DP I is involvedin replacing deoxyribonucleotides after removal of RNA primerson the lagging strand.The sliding clamp is an accessory protein that helps hold theDNA polymerase onto the DNA strand during replication.RNA Primase Is actually part of aggregates of proteins calledprimosomes. This enzyme attaches a small RNA primer to thesingle stranded DNA onto which DP III can adddeoxyribonucleotides.6/15/2013DNA REPLICATION Part-I43
  44. 44. RNase This enzyme dismantles the RNA primerspresent at the 5’ end of each Okazaki fragment onlagging strand and also single RNA primer present onthe leading strand at the 5’ end.DNA ligase Can catalyse the formation ofphosphodiester bond between 3’OH and 5’phosphate groups of two DNA fragments on laggingstrand after removal of RNA primers. They seal thegaps between two such DNA pieces.6/15/2013DNA REPLICATION Part-I44
  45. 45. REPLICATION FORKThe point where the DNA is separated into single strands, andwhere new DNA will be synthesized, is known as the replicationfork.For replication to take place the two strands of DNA doublehelix at weak spots ( Where more A------- T base pairing is present )separate by dissolving Hydrogen bond. The separated strandsappear as bubble under Electron Microscope. The two halves ofthis bubble look as Y shaped Replication Fork with Neck and Mouth.In prokaryotes there is single origin (Ori) , where as in Eukaryotesseveral Ori are present.6/15/2013DNA REPLICATION Part-I45
  46. 46. 6/15/2013DNA REPLICATION Part-I46REPLICATION FORK
  47. 47. DNA replication“It has not escaped ournotice that the specificpairing we have postulatedimmediately suggests apossible copying mechanismfor the genetic material.”James WatsonFrancis Crick1953

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