Biochemistry 441 Lecture

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Biochemistry 441 Lecture 2Ted YoungJanuary 6,
2006The utility of complementarity

• Interactions between bases
• Denaturation and renaturation of DNA
• Enzymatic sequencing of DNA

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Base-pairing in nucleic acids

• Standard Watson-Crick base pairs

Note that for both G/C and A/T bp the hydrogen
bond donors and acceptors shown are the only ones
possible if the positions of the C1 atoms
maintain the geometry of the B helix. Other
pairing can occur in unusual DNA structures-for
example at the ends of chromosomes Hoogsteen
base-paired G-quartets are found.
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Base-pairing in nucleic acids-evidence for
pairing in solution
Infrared spectra of bases- N-H region of the
spectrum.
Only for GC does the IR spectrum change,
implying that the N-H bonds are altered this
change is seen only for standard Watson-Crick
base-pairs G-C and A-T not GT, AC, TC, or
GA.
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Base-pairing in nucleic acids
Only Watson-Crick pairs show strong association
in solution
what about this? We will see later that special
G-G base- pairing plays a special role at the
ends of chromosomes-the telomeres
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Base-stacking plays an important role in
stabilizing double-stranded DNA
Studies of the osmotic pressure, p, of solutions
of free bases demonstrates that they aggregate.
p RTm where Rgas constant, Ttemp,
mmolal concentration-a measure of the number of
molecules in solution.
For substances that aggregate

• fRTm, where fosmotic coefficient (which has a
  value
• between 0 and 1) in other words, the effective
  number of
• molecules in solution is reduced by aggregration,
  and this
• reduces the osmolarity.

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Evidence for base stacking in solution
The reduction in f is not due to H-bonding
between bases since N6,N6 methyl adenine cant
H-bond and shows the greatest reduction in f.
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Denaturation of nucleic acids ds DNA

• The forces that hold the two strands of DNA
  together are all weak forces and therefore the
  two strands can be easily separated.

Common denaturing agents Heat, high pH, and
strong H-bonding agents such as urea and
formamide. ((Why not water?)) Why not low pH?
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Structure of denatured DNA

• Rapidly cooling of denatured DNA leads to
  imperfectly base- paired single strands of DNA.
  The figure illustrates intra-strand base-pairing
  that has normal W/C complementarity and
  orientation.
• Questions to consider
• Would all denatured DNA molecules form the same
  structure?
• Would both strands of a double helix form the
  same structure?
• Would single strands of DNA and RNA of the same
  sequence form the same structure?

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DNA-DNA and DNA-RNA hybridization
Single-stranded nucleic acid molecules that are
completely or even partially complementary will
react with one another via hydrogen-bonding to
form stable double-stranded molecules.
radioactive
heat


(DNA or RNA)
Stability
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EM of partially denatured DNA
Annealing of hybridization of two related
single-stranded DNA molecules could produce a
similar picture the location and size of the
single-stranded regions would identify
the location and extent of sequence divergence.
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Denaturation of nucleic acids ds DNA

• The denaturation or melting of double-stranded
  DNA occurs over a very narrow temperature range,
  indicating that it is a highly cooperative
  process.
• The temperature at the midoint of the melting
  curve is known as its melting temperature or Tm.

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Tm can be used to characterize DNA from different
organisms

• Melting temperature is
• affected by (GC) content, ionic strength, and
  solutes that affect hydrophobic and H-bonding
  interactions. In general, agents that disrupt
  H-binds or base stacking _(increase/decrease)_____
  _
• the Tm. Low ionic strength (dashed curve) _
  _(increases/decreases)______ _______ it.

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DNA sequencing by the Sanger (dideoxy) method
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Automated DNAsequencing allowed the completion
of the human genome sequence faster than expected
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Summary

• Biological properties of DNA derive exclusively
  from its chemical and physical properties
• Chemical properties of DNA derive from the
  properties of its three constituents-phosphate,
  sugar and base
• Determination of the sequence of bases in DNA and
  the ability to synthesize DNA chemically have
  revolutionized how genes are studied