DNA Structure and Replication

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DNA Structure and Replication

• Objectives
• 1. Describe the structure of a nucleotide.
• 2. Explain the process, purpose and molecules
  involved in the replication of DNA.

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Actual Structure of Chain

• 3C of nucleotide 1 binds to phosphate group of
  nucleotide 2 to form sugar phosphate backbone
• Nitrogen bases of opposite chains are paired to
  one another as a result of hydrogen bonding
• only pairs allowed
• A - T (2 H bonds) These are considered
• C - G (3 H bonds) COMPLIMENTARY

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Base Pairing Rules
-Sides run in opposite directions
ANTI-PARALLEL -one side runs 5 3. Other runs
3 5 -10 base pairs per turn of helix
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Why is DNA a good material for storing genetic
information?   A linear sequence of bases has a
high storage capacity a molecule of n bases has
4n combinations just 10 nucleotides long --
410 or 1,048,576 combinations Humans 3.2
x 109 nucleotides long 3 billion base pairs
 
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DNA Replication

• Why replicate (copy)?
• 2 months after Watson and Crick proposed DNA
  structure, they wrote a second paper proposing
  the method of replication (1953)
• Experimental proof took 5 years and was provided
  by Matthew Messelson and Franklin Stahl

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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.
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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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Denaturing and Unwinding

• 1. Helicase enzyme that attaches at origin of
  replication (special site where replication
  begins)
• -one origin in bacteria, several in
  eukaryotes
• Once helicase attaches, single stranded binding
  proteins attach to separate strands and hold them
  straight so complementary strand can be
  synthesized. This creates a replication bubble.

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Priming and Initiation

• Primase places a short chain of RNA nucleotides
  (usually lt10 bp) called a primer at origin of
  replication
• This primer is later replaced with DNA
  nucleotides by DNA polymerase

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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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Continuous and Discontinuous Synthesis

• DNA polymerase enzyme that adds complementary
  base pairs to new strand
• 500 nucleotides/second in bacteria
• 50 nucleotides/second in humans
• Can only add nucleotides to free 3 end of
  growing strand - NEVER to 5 end
• not a problem for 3 5 side (LEADING STRAND).
  This side undergoes CONTINUOUS SYNTHESIS

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• The strand running 5 3 (LAGGING STRAND) is
  synthesized DISCONTINUOUSLY
• as replication fork opens up, short segments of
  the new strand are synthesized. These fragments
  are called OKAZAKI FRAGMENTS
• Another enzyme, LIGASE, joins the fragments into
  a single strand.

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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.

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