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  • DNA Replication

By

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Dr. Hany Srour

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Lecture 8

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DNA MAINTENANCE

  • Mutation rate are extremely low
  • 1 mutation out of 109 nucleotides per generation

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DNA replication

Separation, Base pair

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The Chemistry of DNA replication

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DNA Synthesis by DNA polymerase

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DNA Polymerase

Nucleotide polymerizing enzyme, first discovered in 1957

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DNA replication with two forks

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This Doesn’t Work!

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DNA replication Fork

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DNA Proofreading

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Structures of DNA polymerase during polymerizing and editing

E: exonucleolytic; P: polymerization

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Site-directed mismatch repair in eucaryotes

In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

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DNA PROOFREADING�RNA USUALLY DOESN’T HAVE THIS. WHY?

  • Pairing, correct nucleotide has higher affinity binding to the moving polymerase
  • Un-Paired nucleotide is easier to be off before covalent ligation, even after binding.
  • Exonucleotic proofreading
  • Strand directed mismatch repair

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DNA Primer synthesis

On Lagging strand

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DNA Replication at the Lagging strand

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DNA Ligase

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DNA Helicase

DNA double helix are tightly coupled. High temperature is needed to break them (95oC)

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DNA Binding Protein

SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

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SSB Proteins

DNA

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DNA Clamping Protein

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Cycle of DNA Polymerase/Clamping Protein loading and unloading

At the lagging strand (how about leading strand?)

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Protein machinery for DNA replication

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A Moving Replication

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Structure of the Moving Complex

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DNA winding

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DNA topoisomerase I

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DNA topoisomerase II

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DNA topoisomerase II

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Mammalian replication Fork

(eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is the real polymerase)

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SUMMARY

  • DNA replication 5’->3’
  • DNA proof reading
  • Lagging strand, back-stitching, Okazaki fragment
  • Proteins involved:
  • DNA polymerase, primase
  • DNA helicase and single-strand DNA-binding protein (SSB)
  • DNA ligase, and enzyme to degrade RNA
  • DNA topoisomerases