The Dogma

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The Dogma

• Nucleic acid (DNA/RNA) is importantWhy?
• The central dogma of Molecular Biology
• DNA (genes, chromosomes) begets itself
  (replication), as well as RNA (transcription))
• RNA begets protein (translation)
• Which proteins a cell expresses (and how much),
  dictates what a cell does

2
DNA structure

• DNA an ideal molecule for storage of
  information.
• Made of simple, stable(?) bits of information
  (the nucleotide) (metaphor letters)
• Easily assembled/disassembled (metabolism)
  (metaphor words, sentences, books)
• The information is easily read (replication,
  transcription)

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The nucleotide Pentose sugar
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1
Pentose Sugar (2 OHribose, 2Hdeoxyribose)
4
The nucleotide Nitrogenous bases
Pyrimidines (small)
Purines (BIG)
From Kimballs biology pages http//users.rcn.com
/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.h
tml
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The nucleotide
Phosphate
Base
Sugar (2 OHribose, 2Hdeoxyribose)
Nucleotide sugarphosphatebase
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The chain has polarity
DNA chains are connected by a phosphate between
sugar carbons
The chain has polarity the phosphate bridges
between 5 and 3 carbons (almost never 5 and
5, or 3 and 3)
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DNA metabolism

• Making phosphodiester bonds
• Synthesis (Nucleotide addition, nucleotide by
  nucleotide)
• Ligation (joining two polynucleotide chains
  together)
• Breaking phosphodiester bonds.
• Cleavage, or hydrolysis

8
Synthesis
Chemistry dictates addition is always to 3 end
of chain. In other words synthesis is always 5
to 3
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DNA synthesis

• Synthesis requires...
• Substrates
• 3 OH of existing chain (primer strand)
• template strand (see replication lecture)
• deoxynucleotide triphosphate (dNTP)
• Cofactors
• Mg2 (metal cofactor)
• Enzyme (DNA polymerase)
• Products are
• Chain that is longer by one nucleotide
• Pyrophosphate (PPi)

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Cleavage Exonuclease
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Cleavage (hydrolysis)

• Chain is broken between phosphate and sugar (5
  carbon usually retains phosphate)
• Requires.
• Substrate
• DNA chain, usually double stranded
• Water
• Enzyme (nuclease)
• Co-factors usually Mg2
• Product broken chain
• If chain broken from end, enzyme is exonuclease
• Exonucleases can chew from 3 end (3 to 5 exo)
  or 5 end (5 to 3 exo)
• If chain broken in middle, enzyme is endonuclease

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Cleavage Endonuclease
Restriction enzymes are endonucleases (see Lee
lecture)
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Ligation
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Ligation

• Requires
• Substrates
• two DNA chains
• ATP
• Cofactors
• Mg2 (metal cofactor)
• Enzyme ligase
• Products are
• Two chains joined together into one chain
• AMP
• Pyrophosphate (PPi)

15
DNA chains form helices

• Single DNA chains will form a helix (spiraling
  line like threads on screw) because of.
• Hydrophobic interactions between bases
• Bases are carbon rich rings that hide from water,
  and therefore stack on top of each other
• Ionic interactions
• Phosphates are highly negatively charged, thus
  repel each other

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The double helix

• A single stranded DNA chain will form a helix
  but
• Each base has a number of hydrogen donors and
  acceptors
• Donors like to form hydrogen bonds with acceptors
  
• Like this..

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Watson-Crick base pairs

• A with T
• G pairs with C
• Why?
• Complementary pattern of hydrogen donors and
  acceptors
• GC stronger than AT

• From http//users.rcn.com/jkimball.ma.ultranet/Bio
  logyPages/B/BasePairing.html

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The double helix
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The double helix

• Two chains with extended sequence that can pair
  together, or is complementary, may form a
  double helix
• Constraints of backbone structure permits double
  helices only when complementary sequence is of
  the opposite polarity.
• i.e.5GGTCA3 will pair with 5TGACC3, but NOT
  5CCAGT3

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The double helix
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Association/disassociation of the double helix

• Hydrogen bonds between paired bases are weak
• Sensitive to temperature, salt concentration
• Heating will separate, denature, or melt a double
  helix into two separate stands (single stranded,
  or ssDNA)
• Denaturation occurs at a specific temperature
  (melting temperature, or Tm)
• Tm defined by length (longer comlementary
  sequencehigher Tm) and sequence (higher
  GChigher Tm)
• Separation of strands required for replication,
  transcription
• instead of heat, these processes use ATP for
  energy to break base pairs

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Secondary structure
Secondary structure
hairpins (intra-molecular pairing of single
strand) heteroduplex (double stranded DNA with
the occasional mismatch, forms bubbles in
double helix. Can be caused by renaturation of
partially complementary sequence, or replication
errors
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Helical shape

• Helical parameters
• Screw sense left handed, or right handed
• Twist degrees rotation, along the horizontal
  axis, between successive base pairs
• Rise elevation, along the vertical axis, between
  successive base pairs
• Tilt degrees of inclination of base pair from
  the horizontal access (in most double helices
  base pairs are not significantly tilted )

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Helical forms

• A form
• Formed in DNA under dehydrating conditions
• Major form of RNA double helix
• B form
• Standard DNA double helix
• Z DNA
• Forms in vitro primarily at GC rich regions

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Forms of the double helix
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Helical forms

• A form
• Shorter, fatter than B form DNA
• High degree of base pair tilt
• B form
• Standard DNA double helix
• About 34o twist, 3.4 angstrom rise, very little
  tilt
• Z DNA
• Only left handed helix
• Kinked backbone (does not smoothly conform to
  helical shape)
• Much greater rise, reduced twist relative to B DNA

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Forms of the double helix