August 31

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August 31

• DNA replication introduction
• The perfect experiment
• More DNA replication topology, requirements for
  replication, enzymes
• Tuesday finish DNA replication and methods day
• Purifying and working with DNA, gels, DNA
  sequencing, PCR

Thursday will be quiz 1
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DNA is autocatalytic and heterocatalytic One way
flow of information
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DNA replication 1 error/billion bases replicated
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• The DNA Error rate
• 5-3 polymerization 1 x 105
• 3-5 exonucleolytic proofreading 1 x 102
• Strand-directed mismatch repair 1 x 102
• Total 1 x 109

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The parts The whole How do you replicate it?
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Human genome 3 billion base pairs Each
chromosome Average 130 million base pairs E.
coli K12 chromosome 4,639,221 nucleotides
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3 models Conservative Semi-conservative Dispersed
Watson-Crick predicted semi-conservative
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Meselson-Stahl Invented CsCl gradients Used
15N Classic The most beautiful experiment in
biology. John Cairns
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Thinking about models

• Einstein Everything should be as simple as
  possible, but not simpler.
• Ockhams razor The simpler the model the more
  likely it is correct, ie dont go through
  contortions to make a model fit a data set.

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A little bit about DNA topology
Relaxed Supercoiled Twist Writhe Linking number
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The replication fork
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DNA replication problem 1

• Keeping DNA relaxed and accessible

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Unwinding the DNA helicase
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SSB protein
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Topoisomerase I- nicking
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Topoisomerase I- nicking
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Topoisomerase II double-strand passing
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Topoisomerase II double strand passing
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Problem 2-The copier How does it copy the
template?
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The copier is an enzyme

• What is an enzyme?
• How does it work?
• Will the reaction work spontaneously?

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DNA polymerase Free 3OH of a paired base
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Problem 3 A primer Comes from RNA
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Problem 4 Synthesis of the leading
strand Synthesis of the lagging strand
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Lagging strand-Okazaki fragments
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Key terms in DNA Replication
Origin(s) Replication fork Leading and lagging
strand Okazaki fragments Priming, 3OH
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Problem 5 Sealing the lagging strand
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