I' The DNA Double Helix

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I. The DNA Double Helix

• DNA structure
• Double helical
• major, minor grooves
• right-handed
• bases are 3.4 Å apart (10 Å 1 nm)
• 10 bases/turn
• Complementary Base Pairing
• through H bonds AT, G?C
• Antiparallel Strands
• 5 to 3
• 3 to 5

Discussion of original paper in class Friday
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Right- and Left-handed DNA
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Base-Pairing in DNA
AT
G?C
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Structure of RNA
Sugar ribose, not 2-deoxyribose Bases
uracil, not thymine Organization
single-stranded, not double-stranded
How is genetic information in DNA expressed?
First step is transcribing RNA from DNA -
single-stranded RNA is generated using DNA as a
template
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Reading DNA Strands
Single strand of DNA 5-AGCATTCG-3 3-TCGTAAGC-
5 Complementary strand of above, usually written
5 to 3 5-CGAATGCT-3 Double-stranded
fragment is written 5-AGCATTCG-3 3-TCGTAAGC-5
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Learning Check
The sequence of the dwarf gene in garden peas is
as follows
5 - A G C T A C G T -3 3 - T C G A T G C A -5
Write the RNA sequence transcribed from the top
strand of DNA, 5- 3.
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II. Analytical analyses of nucleic acids
Denaturation/Renaturation
Determining the Tm allows for an estimate of the
base composition of a DNA sample
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2
Which DNA has higher GC content and why?
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Nucleic Acid Hybridization
Transcription of 1 strand of DNA 3
G G T T G G G C C A A C C C
A C G C T T G C G A
2
1
U U U G C G C
T T T G C G C A AA C G C G
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Add RNA to denatured DNA allow to hybridize
Heat - denature
A C G C T T G C G A
G G T T G G G
G G T T G G G C C A A C C C
A C G C T
2
1
C C A A C C C
T G C G A
U U U G C G C
Hybrid
T T T G C G C
A A A C G C G
3
A AA C G C G
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Nucleic Acid Gel Electrophoresis
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What makes nucleic acids acidic?
Base Pairing Rules
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Points to know about DNA structure

• Note how many hydrogen bonds are in the base
  pairing
• If 2, then the pair is AT
• If 3, then the pair is GC
• Recall that A and G are purines with 2 rings,
  while T and C are pyrimidines with 1 ring also T
  has a CH3 group on its ring.

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III. DNA Replication
How is genetic information replicated accurately
at each cell division? Could each strand of the
DNA double helix act as a template for the
complementary strand?
At each cell division, 109 base pairs are
replicated. If error rate is 10-6 , then 3000
errors/cell division - TOO many.
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DNA Replication is Semiconservative
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Other Theoretical Possibilities
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Separation of Nucleic Acids by CeCl Gradient
Centrifugation
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Meselson-Stahl Experiment
DNA Labeling with 15N
Subsequent Generations Labeled with 14N
Cesium Chloride Gradient Banding
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Expected Results From Conservative or Dispersive
Reproduction
If Conservative Two bands, heavy and light, in
1st and 2nd generations
If Dispersive, one smeary band in 1st and
2nd generations
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Expected Results if Semiconservative
These results were obtained. A related
experiment was performed in plants (Fig. 12.5)
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Bacterial DNA Replication begins at a Single
Origin and Proceeds Bidirectionally
Origin of Replication
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DNA Polymerase I can Synthesize DNA

• Arthur Kornberg et al. (1957) discovered the
  enzyme in E. coli
• Requires template DNA strand, primer, MgCl2, and
  4 dNTPs
• Monomers added 5 to 3

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5 to 3 Addition of Monomers
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DNA polymerases I, II and III

• pol I
• most abundant (400/cell)
• RNA primer removal
• pol II
• unknown abundance
• DNA repair?
• pol III
• low abundance (15/cell)
• DNA replication

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Problems of DNA Synthesis

• Unwinding
• Tension must be relieved
• Priming
• Antiparallel strands
• RNA primer removal
• Backbone joining
• Proofreading

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Steps of DNA Synthesis

• Denaturation and Unwinding
• Priming and Initiation
• Continuous and Discontinuous Synthesis
• Including Proofreading and Error Correction
• Removal of Primer
• Ligation of nicks in backbone

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Steps of DNA SynthesisDenaturation and
Unwinding of DNA

• DnaA, DnaB, DnaC proteins are helicases which
  bind origin and separate strands
• Single-strand binding protein (SSBP) keeps
  strands apart
• DNA gyrase, a type of DNA topoisomerase, cuts to
  relax supercoiling

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Initiation of Synthesis

• RNA Primase makes RNA primer on DNA template
• DNA Polymerase III extends primer with DNA
• DNA Polymerase I removes RNA primer, replaces
  with DNA

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Directionality of DNA synthesis
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Proofreading occurs as polymerase moves along
if incorrect base pairing, base is removed and
replaced.
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Continuous and Discontinuous Synthesis

• Continuous
• on Leading Strand.
• Discontinuous
• on Lagging Strand
• creates Okazaki
• fragments.
• DNA ligase joins
• nicks in backbone.