DNA Replication and Repair

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• DNA Replication and Repair

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Watson and Crick Predicted Semi-conservative
Replication of DNA

• Watson and Crick "It has not escaped our notice
  that the specific (base) pairing we have
  postulated immediately suggests a possible
  copying mechanism for the genetic material."
• The mechanism Strand separation, followed by
  copying of each strand.
• Each separated strand acts as a template for the
  synthesis of a new complementary strand.

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The Semiconservative Model

• Matthew Meselson and Franklin Stahl tested
  semi-conservative model
• Template DNA labeled with 15N nucleotides. (more
  dense than normal DNA)
• Fed 14N nucleotides. (newly synthesized DNA was
  less dense than template)
• Isolated DNA at different times and fractionated
  DNA on a density gradient
• denser/heavier DNA found lower in the gradient.
• Less dense/lighter DNA found higher in gradient.

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Replication is bidirectional
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• E. coli genome size 4.6 X 106 bp
• Bacteria have circular chromosome with single
  origin of replication.
• Replication rate is 1000 base pairs per second.
• Duplicate chromosome in 38 minutes.

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• Eukaryotes have larger genomes 3 X 109 bps
• Rate of Eukaryote chromosome replication is
  slower
• But because eukaryote chromosomes have multiple
  origins of replication, it takes about the same
  amount of time to replicate complete genome.

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DNA Replication is Semidiscontinuous
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Okazaki Fragments
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The Enzymology of DNA Replication

• If Watson and Crick were right, then there should
  be an enzyme that makes DNA copies from a DNA
  template
• In 1957, Arthur Kornberg and colleagues
  demonstrated the existence of a DNA polymerase -
• Three DNA polymerases in E. coli
• DNA polymerase I DNA repair and participates in
  synthesis of lagging strand
• DNA polymerase II DNA repair
• DNA polymerase III major polymerase involved in
  DNA replication.

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DNA Polymerase II is a Multisubunit Enzyme
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DNA Polymerase II Subunit Organization
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DNA Replication is a Processive Process.

• DNA Polymerase remains bound to the replication
  fork.
• Dimer of b-subunit forms ring structure around
  the growing DNA chains.

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DNA Polymerase also has proof reading function

• The polymerization reactions have an error rate
  of 1 mistake for every 100,000 base pairs
  incorporated (1 X 10-5 errors per base)
• DNA polymerase has 3 to 5 exonuclease function
  (epsilon-subunit) that recognizes base pair
  mismatches and removes them.
• Therefore proof reading function helps eliminate
  errors which could lead to detrimental mutations.
• However proof reading exonuclease has error rate
  of 1 mistake for every 100 base pairs (1 X 10-2
  errors per base)
• Overall error rate is 1 X 10-7 errors per base.

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Stages of DNA Replication

• Initiation
• Elongation
• Termination

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Initiation of Replication

• in E. coli
• The replisome consists of DNA-unwinding
  proteins, the priming complex (primosome) and two
  equivalents of DNA
• polymerase III holoenzyme
• Initiation DnaA protein binds to repeats in ori,
  initiating strand separation and DnaB, a helicase
  delivered by DnaC, further unwinds. Primase then
  binds and constructs the RNA primer

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Elongation Stage of Replication

• Elongation involves DnaB helicase unwinding, SSB
  binding to keep strands separated.
• Primase Complex Synthesizes short RNA primers.
• DNA polymerase grinding away on both strands
• Topoisomerase II (DNA gyrase) relieves
  supercoiling that remains

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DNA Polymerase I/ Ligase Required to Join Okazaki
Fragments

• DNA polymerase I has 5 to 3 exonuclease
  activity that removes RNA primer.
• Also has 5 to 3 DNA polymerase activity to fill
  in the gap. (proofreading 3-5 exonuclease
  activity)
• Ligase connects loose ends. Used NAD in
  phosphoryltransfer reaction, not a redox reaction
  (Page 643)

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Termination of Replication

• Termination occurs at ter region of E. coli
  chromosome.
• ter region rich in Gs and Ts, signals the end of
  replication.
• Terminator utilization substance (Tus) binds to
  ter region.
• Tus prevents replication fork from passing by
  inhibiting helicase activity.

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DNA Replication in Eukaryotes

• Occurs similarly to what occurs in prokaryotes.
• Multiple origins of replication
• Replication is slower than in prokaryotes.
• 5 different DNA polymerases in Eukaryotes.

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Eukaryotic DNA Polymerases

• Alpha Primer synthesis and DNA repair
• Beta DNA repair
• Gamma Mitochondrial DNA replication
• Delta Leading and lagging strand synthesis, and
  DNA repair
• Epsilon Repair and gap filling on lagging
  strand.

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PCNA analogous to E. coli b-subunit of E. coli
DNA polymerase

• Proliferating cell nuclear antigen
• Trimeric protein
• Sliding clamp structure binds to newly
  synthesized DNA strand

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DNA Repair

• A fundamental difference from RNA, protein,
  lipid, etc.
• All these others can be replaced, but DNA must be
  preserved
• Cells require a means for repair of missing,
  altered or incorrect bases, bulges due to
  insertion or deletion, UV-induced pyrimidine
  dimers, strand breaks or cross-links
• Two principal mechanisms methods for reversing
  chemical damage and excision repair.

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Repair of UV Induced Thymine Dimers
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• Excision-repair systems scan DNA duplexes for
  mismatched bases, excise the mispaired region and
  replace it

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Repair of damage resulting from the deamination
of cytosine

• Deamination of cytosine to uracil is one of most
  common forms of DNA damage
• DNA glycosylases cleave bases at N-glycosidic
  linkages. Leaving sugar-phosphate backbone.
• Endonuclease identifies abscent base and sugar
  phosphate.
• Gap then filled in by DNA polymerase and ligase.