Midterm #2: DNA Replication Flashcards Preview

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Flashcards in Midterm #2: DNA Replication Deck (33):

Flow of Genetic Information

  • DNA Replication: refers to the synthesis of new DNA using the exisiting DNA as a template
  • Transcription: refers to the synthesis of RNA using a gene sequence as a template
  • Translation: refers to the synthesis of protein using mRNA as a template 
  • Note that:
    • Most human DNA is never transcribed
    • not all RNA molecules are translated
    • RNA is sometimes reverse transcribed back to DNA


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Function of DNA

storgage of genetic information 


Define genes

DNA sequences that specify the kinds of proteins made by cells


What does complementary base pairing allow for

  • AT, GC
  • ensure redundancy
  • allows for genetic information to be copied

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DNA polymerase

  • ​carries out DNA replication
  • step-by-step addition of deoxynucleoside monophosphates to a DNA chain (primer strand)

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Reaction of DNA polymerases

  • nucleophilic attack by the 3'-hydroxy group of the primer strand on the alpha-phosphate of the incoming deoxynucleoside triphosphate.
  • This results in the formation of a covalent phosphodiester bond in the transesterification reaction
  • Note that polymerization proceeds in the 5' to 3' direction 
  • A driving force is the subsequent hydrolysis of PPi by inorganic pyrophosphatase 

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All DNA Polymerases Require:

  • Template strand
  • Primer strand with free 3' OH
  • 2'-deoxynucleoside 5'-triphosphates
  • Mg2+


Prokaryotic DNA polymerase I 

  • First characterized is DNA I from E. coli
  • single 103 kDa polypeptide
  • Not essential for DNA replication, but important in DNA repair 

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Pol I has 3 distinct catalytic activities

  • Replication Function (DNA polymerase activity)
    • Moderate processivity (adds 20 bp per encounter with DNA)
    • Moderate rate (25 bp/sec)
    • Moderate fidelity (1 error in 105 bp=10-5error/bp)
  • Editing Function (Exonuclease Acitivty 1, 3'→5')
    • boosts fidelity to 10-8error/bp
  • Repair Function  (Exonuclease Activity 2, 5'→3')

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DNA polymerase III

  • replicates DNA for cell replication
  • Large, multiprotein complex composed of 8 different subunits
    • Very fast: ~1000 bp/sec
    • Very processive: 1000 bp/encounter
    • Possess 3'→5' exonuclease activity, but not 5'→3'
    • polymerase fidelity: ~10-5 error/bp, boosted to 10-7 to 10-8 by exonuclease proofreading

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Initiation Phase of DNA Replication in E. coli 

  • begins at discrete site known as the origin of replication (in E. coli known as oriC)
  • Binding of dnaA protein to the oriC results in unwinding of the DNA duplex
  • The duplex is further unwound with the binding of dnaB and dnaC proteins
  • The unwound DNA is stabalized and protected by single stranded binding protein (SSB)
  • A bubble forms at the oriC with the unwinding of DNA in both directions 

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DNA Replication: Priming Step

  • Primase is an RNA polymerase that binds to single-stranded DNA in a multiprotein complex known as a primosome (7 proteins)
  • Primase synthesizes short (~5-10 base) RNA primers
  • Primase dissociates and Pol III utilizes these RNA primers for the synthesis of DNA 

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DNA Replication: Elongation Phase

  • DNA pol III assembled onto DNA to form the replication fork 
  • Polymerase error rate 10-5
  • 3'→5' exonuclease corrects >99% to make 10-7 to 10-8 error rate 

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Leading vs. Lagging strand, supercoiling

  • Leading Strand
    • template strand is oriented 3'→5'
  • Lagging Strand
    • template strand is oriented 5'→'3
    • Loops out during replication
    • Okazaki fragments
  • Positive supercoiling from unwinding DNA at replication fork is undone by DNA gyrase
    • Induces negative supercoils as it hydrolyzed ATP

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Termination of DNA Replication

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  • Replication of E. coli genome ends when replication forks meet at the termination region (ter)
  • The "concatanes" are resolved  by topoisomerase IV

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Fluroquinolone antibiotics

  • Ciprofloxacin, Levofloxacin
  • Inhibit both DNA gyrase (gram negative) and topo IV (gram positive) enzymes 


DNA Mismatch Repair: Overview

  • This system fixes >99% of the rare errors made by DNA pol, boosting the overall fidelity to 10-9 to 10-10 error/bp
  • E. coli genome: ~5x106 bp
  • Human genome: ~3x109 bp


Mismatch Repain: Features

  • MutS recognizes mismatch
  • Recruits MutL and MutH
  • MutH nicks the replicated DNA
  • UvrD helicase comes in and unwinds DNA
  • Exonuclease removes DNA
  • DNA pol holoenzyme fixes mistake
  • Ligase ligates the strands together
  • ATP is required

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DNA Damage Repair:

  • UV lights, X-Rays, ROS, plant toxins, chemical carcingoens, and therapeutic alkylating agents can damage DNA
  • Base Excision Repair
  • Nucleotide Excision Repair


Base Excision Repair

  • DNA glycosylate removes damaged base from AP site (example, recognizes uracil in DNA)
  • AP endonuclease removes some bases from the middle of the DNA
  • DNA pol I puts in the DNA bases
  • Ligase ligates the strand back together

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Nucleotide Excision Repair

  • Distortions in DNA
  • Uvr-A, Uvr-B, and Uvr-C excise out an area including the damage
  • DNA pol and Ligase

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Bacterial Plasmids

  • Circular, extra-chromosomal, double-stranded DNA molecules that replicate autonomously
    • the number of plasmid molecules per cell is determined by the "strength" of the replication origin (copy number concept)
  • Plasmids may be transferred between bacteria and often carry genes responsible for antibiotic resistance and toxins


DNA Replication in Eukaryotes: The players

  • 13 different eukaryotic DNA pol (all with Greek letters)
  • DNA pol alpha:primase complex synthesizes RNA primer and then adds ~15 bases of DNA. This primer is removed by  FEN-1 nuclease
  • DNA pol delta and epsilon are the main DNA pol involved in chromosomal replication
  • Many other polymerases appear to function primarily in DNA repair pathways (ex: pol beta), plus those found in mitochondria (pol gamma) and chloroplasts


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Topoisomerase Poisons

  • Etoposide (topo II)  and Camptothecin (topo I)
  • Trap topo:DNA complexes and ultimately causes single and double stranded DNA breaks in the duplex
  • Used in cancer chemotherapy