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DNA Replication in eukaryotes and prokaryotes

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Genetic Information Transfer
Central dogma
DNA Replication
General Concepts of DNA Replication

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DNA Replication in eukaryotes and prokaryotes

  1. 1. Genetic Information Transfer 1
  2. 2. Central dogma replication transcription DNA translation RNA protein reverse transcription 2
  3. 3. • Replication: synthesis of daughter DNA from parental DNA • Transcription: synthesis of RNA using DNA as the template • Translation: protein synthesis using mRNA molecules as the template • Reverse transcription: synthesis of DNA using RNA as the template 3
  4. 4. DNA Replication 4
  5. 5. Section 1 General Concepts of DNA Replication
  6. 6. DNA replication • A reaction in which daughter DNAs are synthesized using the parental DNAs as the template. • Transferring the genetic information to the descendant generation with a high fidelity replication parental DNA daughter DNA6
  7. 7. Daughter strand synthesis • Chemical formulation: • The nature of DNA replication is a series of 3´- 5´phosphodiester bond formation catalyzed by a group of enzymes. 7
  8. 8. The DNA backbone • Putting the DNA backbone together – refer to the 3′ and 5′ ends of the DNA PO4 5′ CH2 4′ base O 1′ C 3′ O – O P O O 5′ CH2 2′ base O 4′ 1′ 3′ OH 2′
  9. 9. Phosphodiester bond formation 9
  10. 10. DNA replication system Template: double stranded DNA Substrate: dNTP Primer: short RNA fragment with a free 3´-OH end Enzyme: DNA-dependent DNA polymerase (DDDP), other enzymes, protein factor 10
  11. 11. Characteristics of replication  Semi-conservative replication  Bidirectional replication  Semi-continuous replication  High fidelity 11
  12. 12. §1.1 Semi-Conservative Replication 12
  13. 13. Semiconservative replication Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication 13
  14. 14. Semiconservative replication 14
  15. 15. Experiment of DNA semiconservative replication "Heavy" DNA(15N) grow in 14N medium The first generation grow in 14N medium The second generation 15
  16. 16. Significance The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity. 16
  17. 17. §1.2 Bidirectional Replication • Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand. • The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork. 17
  18. 18. Replication fork 5' 3' 3' 5' 5' 3' 5' direction of replication 3' 18
  19. 19. Bidirectional replication • Once the dsDNA is opened at the origin, two replication forks are formed spontaneously. • These two replication forks move in opposite directions as the syntheses continue. 19
  20. 20. Bidirectional replication 20
  21. 21. Replication of prokaryotes The replication process starts from the origin, and proceeds in two opposite directions. It is named θ replication. 21
  22. 22. Replication of eukaryotes • Chromosomes of eukaryotes have multiple origins. • The space between two adjacent origins is called the replicon, a functional unit of replication. 22
  23. 23. origins of DNA replication (every ~150 kb) 23
  24. 24. §1.3 Semi-continuous Replication The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end. 24
  25. 25. Leading strand On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand. 3' 5' 3' 3' direction of unwinding 5' 5' 25
  26. 26. Semi-continuous replication 26
  27. 27. Okazaki fragments • Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes. • The daughter strand consisting of Okazaki fragments is called the lagging strand. 27
  28. 28. Semi-continuous replication Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication. 28
  29. 29. Section 2 Enzymology of DNA Replication
  30. 30. Enzymes and protein factors protein Mr # function Dna A protein 50,000 1 recognize origin Dna B protein 300,000 6 open dsDNA Dna C protein 29,000 1 assist Dna B binding DNA pol Elongate the DNA strands Dna G protein 60,000 1 synthesize RNA primer SSB 75,600 4 single-strand binding DNA topoisomerase 400,000 4 release supercoil constraint 30
  31. 31. §2.1 DNA Polymerase DNA-pol of prokaryotes • The first DNAdependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology or medicine in 1959. 31
  32. 32. • Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line. • All of them possess the following biological activity. 1. 5′→3′ polymerizing 2. exonuclease 32
  33. 33. DNA-pol of E. coli 33
  34. 34. DNA-pol I • Mainly responsible for proofreading and filling the gaps, repairing DNA damage 34
  35. 35. Klenow fragment N end DNA-pol Ⅰ C end caroid • small fragment (323 AA): having 5´→3´ exonuclease activity • large fragment (604 AA): called Klenow fragment, having DNA polymerization and 3´→5´exonuclease activity 35
  36. 36. DNA-pol II • Temporary functional when DNA-pol I and DNA-pol III are not functional • Still capable for doing synthesis on the damaged template • Participating in DNA repairing 36
  37. 37. DNA-pol III • A heterodimer enzyme composed of ten different subunits • Having the highest polymerization activity (105 nt/min) • The true enzyme responsible for the elongation process 37
  38. 38. Structure of DNA-pol III α : has 5´→ 3´ polymerizing activity ε : has 3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity. θ: maintain heterodimer structure 38
  39. 39. 39
  40. 40. 40
  41. 41. DNA-pol of eukaryotes DNA-pol α: initiate replication and synthesize primers DNA-pol β: replication with low fidelity DnaG, primase repairing DNA-pol γ: polymerization in mitochondria DNA-pol δ: elongation DNA-pol III DNA-pol ε: proofreading and filling gap DNA-pol I 41
  42. 42. §2.2 Primase • Also called DnaG • Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template. • Primers are short RNA fragments of a several decades of nucleotides long. 42
  43. 43. 43
  44. 44. • Primers provide free 3´-OH groups to react with the α-P atom of dNTP to form phosphoester bonds. • Primase, DnaB, DnaC and an origin form a primosome complex at the initiation phase. 44
  45. 45. §2.3 Helicase • Also referred to as DnaB. • It opens the double strand DNA with consuming ATP. • The opening process with the assistance of DnaA and DnaC 45
  46. 46. §2.4 SSB protein • Stand for single strand DNA binding protein • SSB protein maintains the DNA template in the single strand form in order to • prevent the dsDNA formation; • protect the vulnerable ssDNA from nucleases. 46
  47. 47. §2.5 Topoisomerase • Opening the dsDNA will create supercoil ahead of replication forks. • The supercoil constraint needs to be released by topoisomerases. 47
  48. 48. 48
  49. 49. • The interconversion of topoisomers of dsDNA is catalyzed by a topoisomerase in a three-step process: • Cleavage of one or both strands of DNA • Passage of a segment of DNA through this break • Resealing of the DNA break 49
  50. 50. Topoisomerase I (topo I) • Also called ω-protein in prokaryotes. • It cuts a phosphoester bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut. 50
  51. 51. Topoisomerase II (topo II) • It is named gyrase in prokaryotes. • It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds. • It can change dsDNA into the negative supercoil state with consumption of ATP. 51
  52. 52. 52
  53. 53. §2.6 DNA Ligase 3' 5' 5' 3' RNAase 3' 5' OH dNTP P DNA polymerase 3' P 5' ATP 3' 5' 5' 3' 5' 3' DNA ligase 5' 3' 53
  54. 54. • Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand. • Sealing the nick in the process of replication, repairing, recombination, and splicing. 54
  55. 55. §2.7 Replication Fidelity • Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer. • Enzymes use two mechanisms to ensure the replication fidelity. – Proofreading and real-time correction – Base selection 55
  56. 56. Proofreading and correction • DNA-pol I has the function to correct the mismatched nucleotides. • It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication. 56
  57. 57. Exonuclease functions 5´→3´ exonuclease activity cut primer or excise mutated segment 5' 3' 3´→5´ exonuclease activity excise mismatched nuleotides C T T C A G G A 3' G A A G T C C G G C G 5' 57
  58. 58. Section 3 DNA Replication Process
  59. 59. Sequential actions • Initiation: recognize the starting point, separate dsDNA, primer synthesis, … • Elongation: add dNTPs to the existing strand, form phosphoester bonds, correct the mismatch bases, extending the DNA strand, … • Termination: stop the replication 59
  60. 60. §3.1 Replication of prokaryotes a. Initiation • The replication starts at a particular point called origin. • The origin of E. coli, ori C, is at the location of 82. • The structure of the origin is 248 bp long and AT-rich. 60
  61. 61. Genome of E. coli 61
  62. 62. Structure of ori C • Three 13 bp consensus sequences • Two pairs of anti-consensus repeats 62
  63. 63. Formation of preprimosome 63
  64. 64. Formation of replication fork • DnaA recognizes ori C. • DnaB and DnaC join the DNA-DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space. • DnaA is replaced gradually. • SSB protein binds the complex to stabilize ssDNA. 64
  65. 65. Primer synthesis • Primase joins and forms a complex called primosome. • Primase starts the synthesis of primers on the ssDNA template using NTP as the substrates in the 5´- 3´ direction at the expense of ATP. • The short RNA fragments provide free 3´-OH groups for DNA elongation. 65
  66. 66. Releasing supercoil constraint • The supercoil constraints are generated ahead of the replication forks. • Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint. • The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA. 66
  67. 67. Primosome complex Dna B Dna A Dna C primase 3' 5' 3' DNA topomerase 5' 67
  68. 68. b. Elongation • dNTPs are continuously connected to the primer or the nascent DNA chain by DNA-pol III. • The core enzymes (α 、、 and θ ) catalyze the synthesis of leading and lagging strands, respectively. • The nature of the chain elongation is the series formation of the phosphodiester bonds. 68
  69. 69. 69
  70. 70. • The synthesis direction of the leading strand is the same as that of the replication fork. • The synthesis direction of the latest Okazaki fragment is also the same as that of the replication fork. 70
  71. 71. 71
  72. 72. Lagging strand synthesis • Primers on Okazaki fragments are digested by RNase. • The gaps are filled by DNA-pol I in the 5´→3´direction. • The nick between the 5´end of one fragment and the 3´end of the next fragment is sealed by ligase. 72
  73. 73. 3' 5' 5' 3' RNAase 3' 5' OH dNTP P DNA polymerase 3' P 5' ATP 3' 5' 5' 3' 5' 3' DNA ligase 5' 3' 73
  74. 74. c. Termination • The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32. • All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase. 74
  75. 75. §3.2 Replication of Eukaryotes • DNA replication is closely related with cell cycle. • Multiple origins on one chromosome, and replications are activated in a sequential order rather than simultaneously. 75
  76. 76. Cell cycle 76
  77. 77. Initiation • The eukaryotic origins are shorter than that of E. coli. • Requires DNA-pol α (primase activity) and DNA-pol δ (polymerase activity and helicase activity). • Needs topoisomerase and replication factors (RF) to assist. 77
  78. 78. b. Elongation • DNA replication and nucleosome assembling occur simultaneously. • Overall replication speed is compatible with that of prokaryotes. 78
  79. 79. c. Termination 3' 5' 5' 3' 3' 5' 5' 3' 3' 5' connection of discontinuous segment 5' 3' 3' 5' 5' 3' 79
  80. 80. Telomere • The terminal structure of eukaryotic DNA of chromosomes is called telomere. • Telomere is composed of terminal DNA sequence and protein. • The sequence of typical telomeres is rich in T and G. • The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other. 80
  81. 81. Telomerase • The eukaryotic cells use telomerase to maintain the integrity of DNA telomere. • The telomerase is composed of telomerase RNA telomerase association protein telomerase reverse transcriptase • It is able to synthesize DNA using RNA as the template. 81
  82. 82. 82
  83. 83. Significance of Telomerase • Telomerase may play important roles is cancer cell biology and in cell aging. 83
  84. 84. Section 4 Other Replication Modes
  85. 85. §4.1 Reverse Transcription • The genetic information carrier of some biological systems is ssRNA instead of dsDNA (such as ssRNA viruses). • The information flow is from RNA to DNA, opposite to the normal process. • This special replication mode is called reverse transcription. 85
  86. 86. Viral infection of RNA virus 86
  87. 87. Reverse transcription Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA. 87
  88. 88. Process of Reverse transcription • Synthesis of ssDNA complementary to ssRNA, forming a RNA-DNA hybrid. • Hydrolysis of ssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA. • Synthesis of the second ssDNA using the left ssDNA as the template, forming a DNA-DNA duplex. 88
  89. 89. 89
  90. 90. Reverse transcriptase Reverse transcriptase is the enzyme for the reverse transcription. It has activity of three kinds of enzymes: • RNA-dependent DNA polymerase • RNase • DNA-dependent DNA polymerase 90
  91. 91. Significance of RT • An important discovery in life science and molecular biology • RNA plays a key role just like DNA in the genetic information transfer and gene expression process. • RNA could be the molecule developed earlier than DNA in evolution. • RT is the supplementary to the 91
  92. 92. Significance of RT • This discovery enriches the understanding about the cancercausing theory of viruses. (cancer genes in RT viruses, and HIV having RT function) • Reverse transcriptase has become a extremely important tool in molecular biology to select the target genes. 92
  93. 93. §4.2 Rolling Circle Replication 3' 5' 3' 5' 3' 5' 93
  94. 94. §4.3 D-loop Replication 94

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