Restriction Enzymes

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Restriction Enzymes

• Cleave DNA molecules at specific sites
• Common tools for molecular biology

ATCG
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Restriction Enzymes

• Restriction and modification systems
• A defense mechanism in bacteria

DNA
Restriction
Methylation
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Restriction Enzymes

• Three types of restriction enzymes
• Type I
• recognize a non-symmetric specific sequence, but
  cleave randomly.
• Type II
• recognize a specific and often symmetric sequence
  and cleave at specific sites.
• Type III
• recognize a symmetric sequence, but cleaves
  outside of the recognition site

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Restriction Enzymes

• Type II are most useful in molecular biology
• Example EcoRV
• Recognizes the DNA sequence
  5-AATGCGATATCCGGC-3
• 
  3-TTACGCTATAGGCCG-5

Palindromic sequence
(Cognate sequence)
5-AATGCGAT-3 5-ATCCGGC-3 3-TTACGCTA-5
3-TAGGCCG-5
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Restriction Enzymes

• There are more than 2,500 enzymes that recognize
  over 200 specific sites.

EcoR 1
5' - A A T T C - 3 3'-G - 5'
5' - G A A T T C - 3 3' - C T T A A G - 5'
5' - G 3' - C T T A A- 5'
Cohesive ends vs. blunt ends
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Methylation Prevents Cleavage by Restriction
Enzymes
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Reaction Catalyzed by Restriction Enzymes
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Reaction Catalyzed by Restriction Enzymes
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Two Possible Mechanisms
Mechanism 1
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Two Possible Mechanisms
Mechanism 2
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Two Possible Mechanisms
Common pentacoordinate transition state
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Mechanism for Cleavage of Phosphodiester Backbone
Phosphorothioate
The results prove that mechanism 2 is correct.
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Mg2 is essential
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Palindromic sequence
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(No Transcript)
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C
G
T
A
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Distortion of the DNA in the Site
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Specificity of Restriction Enzymes is not
Determined by Binding Affinity

• Binding of cognate DNA sequence to enzyme induces
  a proper distortion of the DNA for cleavage.
• Binding of nonspecific DNA does not form a
  complete catalytic center including Mg2

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Non-specific and cognate DNA
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Binding Energy of EcoRV to Cognate DNA and
Nonspecific DNA
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Methylation Precludes the Formation of Specific
Interaction between DNA and Enzyme