Eukaryotic chromosomes

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Eukaryotic chromosomes
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DNA structure review

• Complementary antiparallel strands of DNA held
  together by Hydrogen bonds to form a
  double-stranded helix
• Directionality to chain
• 5 phosphate to 3 hydroxyl
• Grows only from 3 end

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DNA Replication

• Semiconservative
• Parent strand dark blue
• Daughter strand light blue

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• Parent strand provides template for antiparallel
  complementary daughter strand

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DNA replication

• Begins at special DNA sequences called
• origins of replication
• ori

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DNA replication

• Begins at ori
• One ori for single circular DNA in prokaryotic
  cells
• Replicates in both directions
• bidirectional

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DNA replication

• Eukaryotic DNA requires multiple ori
• Replication bubbles form at each ori
• Each bubble has two replication forks
• Bubbles fuse --gt strands separate

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Electron Microscopy of replicating DNA
reveals replicating bubbles. How does one
prove bidirectional fork movement?
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• Each replication bubble has two leading strands
  and two lagging strands

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DNA polymerase cannot initiate DNA synthesis

• 5 3
• nucleotides
• DNA polymerase
• No reaction

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DNA polymerase requires a primer

• 5 ' 3 '
• 3 ' 5 ' nucleotides
• DNA polymerase
• primer
• 5 ' 3 '
• 3 ' 5 '

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Replication fork
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Special proteins unwind DNA double helix

• Helicases use ATP to unwind DNA

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DNA polymerases catalyze the elongation of DNA
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Looping the lagging strand to make both
polymerases move in the same direction
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DNA polymerases proofread their work

• Check that last nucleotide is correct
• Correct any errors before adding next nucleotide

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DNA pol I (polA)

• small fragment 5 to 3 exonuclease only. This
  activity allows pol I (intact) to be used for
  nick translation in vitro.

68 kDa
35 kDa
Klenow fragment
small fragment
N
C
exonuclease 3-5
polymerase
exonuclease 5-3
Proofreading domain
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DNA polymerase error rates

• Initial pairing error 1/105
• After proofreading - Final copy 1/109
• Human genome 3.2 x 109 bp
• 3 errors/replication!

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Primase special RNA polymerase required for DNA
replication

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Completion of replication

• Circular chromosome synthesis is bidirectional
• -forms two circles

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Completion of replication

• Linear chromosomes have an end problem
• DNA polymerase cannot fill gap at 3 ' end
  following primer removal

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Completion of replication

• Ends of linear DNA telomeres
• Highly repeated DNA
• Special DNA polymerase (telomerase) adds extra
  repeats

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