Fundamentals I: 11:00 - 12:00Scribe: Christopher Bannon

Thursday, September 10 5, 2009Proof: Jessica Jarvis

Dr. ChesnokovDNA Metabolism: Replication, Recombination and RepairPage1 of 54

  1. DNA Metabolism [S1]:
  2. Essential Question [S2]
  3. –read from slide
  4. Outline [S3]
  5. – The essential question, of how genetic information, that is stored in form of DNA, how the information is replicated, how it is rearranged, and how the integrity of the information is maintained in the face of damage.
  6. What the difference is in the replication process of eukaryotic vs. bacterial cells
  7. Red items = what were covering (read directly off the slide)
  8. Watson and Crick’s Famous Paper [S4]
  9. – Mechanims of DNA replication was proposed by Watson and Crick article called “moleculuar structure of nucleic acids” and it is referred to as “dawn of molecular biology” essentially describing model of DNA double helix
  10. The Dawn of Molecular Biology [S5]
  11. – Noticed and postulated specific base pairing, immediately suggesting possible copying mechanisms for genetic material and this mechanism involves strand sepreration followed by copying of each independent strand. And each separated strand acts as a template for the synthesis of a new compliment strand.
  12. This hypothesis was demonstrated to be true. Te major features of DNA replication which are found in all cells be they bacterial or eukaryotic.
  13. How is DNA Replicated [S6]
  14. – read directly from slide
  15. Untwisting DNA Strands [S7]
  16. –Read off slide
  17. Three Models of DNA Replication… [S8]
  18. – Theoretically 3 possible mechanisms for DNA duplication
  19. Both models (Dispersive, and Conservative) proved to be incorrect
  20. Semiconservative Model of DNA replication proved to be correct. This model involves reassoation of two parent strands and each strand is copied to generate a complimentary strand. As a result each daughter strand contains 1 paretnal and 1 newly synthesized strand….Audio Cuts Out.
  21. The Meselson and Stahl Experiment [S9]
  22. – Audio Cuts Out. …grew bacterial cells in presence of nucleotides which contained muddied bases and those bases contained heavy homologue of nitrogen and by bacterial cells growing in presence they pick up and introduce that homologue into their newly synthesized genetic material. After certain time shift bacterial cells to a new media which contains “light nitrogen” and then you isolate DNA and separate it based on density centrifugation.
  23. Each model, DNA replication would be semi conservative… Audio cuts out.
  24. Newly synthesized strands would contain heavy nitrogen
  25. During density centrifugation you get 2 distinct peaks one corresponds to parental or light DNA and the other corresponds to newly synthesized or “heavy/light” DNA. One strand of newly strand would contain light parental and other is new heavy strand. By keeping this process longer you can actually achieve result of where newly synthesized strand would become completely heavy shown on slide. Shifted towards both heavy/heavy when both daughter strands become heavy when you keep in long enough in the medium containing heavy nucleotides
  26. DNA Replication is Bidirectional [S10]
  27. –2nd important feature of DNA replication is that it is bidirectional. This means that DNA replication initiates at the origin of DNA replication and replication forks corresponding to un
  28. DNA replication initiates at origins of replication and forks correspond to untwisting of parental DNA move in opposite direction. Proved by simple experiments where cells were labeled by a short time with radioactive nucleotides. DNA was isolated and stretched on a glass slide and exposed to autoradiography and after developing of x-ray film, one could see incorporation of radioactively labeled nucleotides in both directions from the origin.
  29. DNA Replication is Semi-Discontinuous [S11]
  30. – The term Semi-continuous means that DNA polymerase (the enzyme which synthesizes DNA) which replicates both strands at once. But they strands are antiparallel. But DNA replication must also go from 5’-3’. So in one strand called the leading strand, synthesis can proceed smoothly in 5’3’ direction, but the other strand has to be synthesized backwards from the replication fork (shown on the slide). This process of reverse transcription results int eh formation of “discontinuous fragments” , which are called Okazaki fragments, and each of these fragments has its own rNA primers and after completion of DNA replication the RNA primers need to be removed and those short fragments have to be ligated together with the assistance of the enzyme DNA ligase.
  31. The Enzymology [S12]
  32. – if model from Watson and crick is correct then there should be an ezyme that makes DNA copies from a DNA template. This enzyme, DNA polymerase, was discovered 4 years after Crick’s proposed theory of the DNA model was accepted as true. The strand that was isolated was termed polymerase I.
  33. IT was shown that this enzyme requires 3’-OH deoxy nucleotides, a template, and a primer which interacts and pairs with a template to forma short double stranded region.
  34. DNA Polymerase I [S13]
  35. – read from slide
  36. Picture of DNA Polymerase I (yellow globular protein) [S14]
  37. –AUDIO CUTS OUT HERE. THE REST OF THIS TRANSCRIPT IS WHAT I WAS ABLE TO WRITE DOWN DURING LECTURE, BUT I CAN’T BE 100% POSITIVE IT IS ALL CORRECT AS I AM NOT ABLE TO CHECK WITH AUDIO FILE. USE IF YOU CARE TO
  38. Shown is a spaced filling model of DNA polymerase I. Nucleotides are added From left
  39. DNA synthesis is catalyzed by DNA Polymerase [S15]
  40. – reaction catalyzed by DNA polymerase, nucleotides are added to 3’ position of growing strand and this reaction is driven by large favorable energy ….read from slide
  41. Polymerizing and Editing [S16]
  42. – structure of DNA polymerase
  43. 2 sites 1 polymerizing and 1 is editing
  44. proof reading function, if incorrect base is added, it is always checked in editing site. The incorrect base can be excised and replaced at the editing site.
  45. POlII does …. On both leading and lagging strand???
  46. DNA Polymerase III [S17]
  47. – beta subunit forms a ring and stabilizes it sp DNA doesn’t fall off during synthesis
  48. Table 28.2: Subunits of E. coli [S18]
  49. – table of subunits and functions went over on last slide , We should probably know these, at least alpha - beta
  50. Ribbon Diagram of the Beta-Subunit… [S19]
  51. – see how it wraps around DNA stabilizing
  52. General Features of the Replication Fork [S20]
  53. – summarize general features of replication fork based primarly on…
  54. all processes are similar in bacteria to eukaryotic cells
  55. DNA gyrase = enzyme which removes/ reduces supercoils in DNA and then the helicase unbinds DNA and then the single stranded DNA binds to a protein. Read from slide
  56. Table 28.3: Proteins involved in DNA Replication… [S21]
  57. – reading table
  58. Features of Replication: Summary [S22]
  59. – mostly based on studies of bacterial cells though process is similar in eukaryotic cells
  60. read off slide
  61. semidiscontinuous = leading strand is formed continuously and lagging strand is formed in segments
  62. The Proteins that Initiation DNA Replication in Bacteria [S23]
  63. – ????? happens later when Primosome, is composed of dnaB (Helicase) and dnaG (DNA primase), joins the complex. Once DNA Helicase inhibitor is removed, DNA Helicase can unwind double helix ????
  64. Possible Mechanism of Replicative Helicase Action [S24]
  65. – Essentially this is evolution of how Helicase might work. C & D are the currently acceptable models of Helicase. When it wraps around double stranded models of DNA and they are physically associated with each other and bumps ???? in unwinding of DNA ;??????? forming a so called replication machinery consisting of dozens of proteins working together.
  66. Eukaryotic DNA Replication [S25]
  67. – Very similar to bacterial DNA but is much more complex b/c there is much more bases to copy. Therefor need more origins of replication, bacteria have only 1 origin of DNA replication.
  68. Read rest from slide
  69. The Phases of the Cell Cycle [S26]
  70. – DNA synthesis – s phase, g2  G stands for gap phase .. get rest from audio
  71. Bacteria Carry their Genes in a Single DNA Molecule [S27]
  72. – human genome consists of 24 different chromosomes. Each consisting of
  73. during DNA synthesis ??????
  74. Eukaryotic Chromosomes Contain Multiple Origins… [S28]
  75. –Experiment proved that eukaryotic chromosomes come from multiple origins of DNA replication
  76. cells were briefly pulsed with radioactive nucleotides
  77. after radiography can see spots of ???? which occurred at multiple places
  78. Conclusions from experiment ???? This seems similar the experiment that was discussed when proving semi-continuous replication process.
  79. Model for Initiation of the DNA Replication Cycle in Eukaryotes [S29]
  80. –ORC binds to initial stages of DNA replication and serves as a platform for complex proteins???
  81. M Late M G1 (Pictorial) [S30]
  82. – still doesn’t include all the proteins which are required for the initiation complex
  83. just wanted to mention that DNA helicase by itself is not enough to initiate DNA replication, what is actually need is CMG complex consisting of about 12 proteins working together during unbinding of DNA double helix show in S on left
  84. Potential Mechanism Controlling ORC…[S31]
  85. – sequences are important for interaction but also it has been shown that DNA replication origins are usually clustered near rings of frequently transcribed genes. ????
  86. local chromatin structure has also shown to be important and uses position of nucleosomes. All of this is several factors which are important in determining???…this guy is ridiculous
  87. Possible Mechanism of Replicative Helicase Loading [S32]
  88. – a and b represent evolution of our understand. What is known now is that 7 subunits act together act as ATPases to open helicase ring to wrap it around DNA and this process is still not determined with a molecular mechanism.
  89. How six membered ring is actually opened??? Not known???
  90. Proteins that Perform Analogous Function at Replication Forks [S33]
  91. – comparison of proteins in mammalian and bacterial cells
  92. sliding clamp is a ???? unit of bacterial DNA and stabilizes …
  93. in mammalian cells it is a completely separate protein
  94. allows to go for millions of bases without reloading
  95. Question ”should we know all these proteins”
  96. Answer “due to compression of lectures , what u have to know there is binding protein, thesis a Helicase, and there is ???
  97. Table 28.4: Biochemical Properties… [S34]
  98. – Mistake in 4th edition
  99. major polymerase, it is actually polymerase “epsilon” not sigma although it can be used, “epsilon” is the prominent one but for our purposes it may not be important he just wanted to mention it
  100. A Mammalian Replication Fork[S35]
  101. – again function of the proteins is pretty much the same. DNA Helicase on the right polymerase stabilized by sliding clap???
  102. Get from audio
  103. How Are the Ends of the Chromosomes Replicated [S36]
  104. – structures at end of chromosome are called telomeres. replication for telomeres is complete different and requires RNA dependent DNA polymerase called telomerase
  105. Telomere Replication [S37]
  106. – mechanism of telomere replication
  107. at end of DNA when this primer is removed, there is no DNA results in a primer gap and end is gradually lost. What happens is then addition of DNA sequences are added to DNA polymerase????
  108. Telomerase Replicates the Ends of Chromosomes [S38]
  109. Cartoon showing structural DNA dn rna polymerase. Contains rRNA protein and this particular rna serves as a template which can allows for extension of
  110. by this enzyme interacting in a complementary manner with the telomere allows it to extend it along RNA ???? which creates a necessary DNA template separation of RNA primer???
  111. That is why length of a chromosome is
  112. Without this enzyme length of chromosome would shorten over time.
  113. A Mammalian Replication Fork [S39]
  114. = video for replication and transcription, we might want to check out

[end 48 min]