Download Bchem 4200 Part13 - U of L Class Index

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 4200
II. Macromolecular Interactions
Structure and Mechanism III
Restriction Endonucleases
Restriction Endonucleases
Their principal biological function is the protection of the host genome
against foreign DNA (in particular bacteriophage DNA)
Restriction endonucleases occure ubiquitously among prokaryotes.
They are part of the restriction-modification (RM) system, which comprises
an endonuclease and a methyl transferase activity,
endonuclease acts on the foreign DNA
(defined recognition sequence)
methyl transferase acts on the host DNA
(defined recognition sequence)
Almost all DNA methylases target the adenosine base.
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Restriction Endonucleases
There are a number of different sub classes restriction endonucleases.
Type I : Recognize specific sequences and cut DNA at
a nonspecific site > than 1,000 bp away.
Type II: Recognize palindromic sequences and cut DNA within
the palindrome.
Type III: Recognize specific 5-7 bp sequences and cut DNA
24-27 bp down stream of the site.
Type II restriction endonucleases are the most used class as they recognize
and cut specific palindromic sequences in DNA → DNA technology
Restriction Endonucleases
There are a number of different sub classes restriction endonucleases.
Type I : Recognize specific sequences and cut DNA at
a nonspecific site > than 1,000 bp away.
is a palindrome
? and cut DNA within
Type II: RecognizeWhat
palindromic
sequences
the palindrome.
Type III: Recognize specific 5-7 bp sequences and cut DNA
24-27 bp down stream of the site.
Type II restriction endonucleases are the most used class as they recognize
and cut specific palindromic sequences in DNA → DNA technology
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What is a Palindrome?
A palindrome is anything that reads the same forwards and backwards:
Mom
Dad
Tarzan raised Desi Arnaz rat.
DNA Palindromes
Because DNA is double stranded and the strands run antiparallel,
palindromes are defined as any double stranded DNA in which
reading 5’ to 3’ both are the same.
• The EcoRI cutting site:
5'-GAATTC-3
3'-CTTAAG-5'
• The HindIII cutting site:
5'-AAGCTT-3'
3'-TTCGAA-5'
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Type II Restriction Endonucleases (orthodox)
The main criterion for classifying a restriction endonuclease as type II:
it cleaves specifically within or close to its recognition site
it does not require ATP or GTP for its nucleolytic activity
it is a homodimer of ~ 2 x 30 kDa molecular mass
it recognizes palindormic sequences of 4-8 bp in length
it requires Mg2+ for its nucleolytic activity
it cleaves the bond between the 3’-OH and the 5’-phosphate
Type II Restriction Endonucleases (orthodox)
These enzymes can generate three different classes of products:
Product name
Example
Products
Blunt ends
EcoRV
5’ … GAT ATC … 3’
3’ … CTA TAC …5’
3’ Sticky ends
EcoRI
5’ … G
AATTC … 3’
3’ … CTTAA
G … 5’
5’ Sticky ends
BglI
5’ … GCC NNNN
NGGC … 3’
3’ … CGG N
NNNNCCG … 5’
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Type II Restriction Endonucleases
Many type II restriction endonucleases do not conform to these narrow
definitions → subdivisions are necessary
Type II Restriction Endonucleases
Structural similarity of the type II restriction endonucleases suggest a
common (although distant) ancestor.
The restriction endonuclease superfamily can be devided in two branches:
The EcoRI Family
bind DNA from
the major groove
produce sticky and
with 5’-overhangs
common core elements
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Type II Restriction Endonucleases
Structural similarity of the type II restriction endonucleases suggest a
common (although distant) ancestor.
The restriction endonuclease superfamily can be devided in two branches:
The EcoRV Family
bind DNA from
the minor groove
produce blunt ends
common core elements
Type II Restriction Endonucleases
Within the comon core only four β-strand are absolutely conserved.
Two of the strands (β2 and β3 in EcoRI and βd and βe in EcoRV)
contain the amino acids residues directly involved in catalysis.
common core elements
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Interaction with DNA
Restriction endonucleases interact with DNA in a complex manner
A minimal scheme for the
nuclease reactions involve
at least five steps
1. non-specific binding to DNA
2. linear diffusion along DNA
-sliding along major groove
-hopping helical turns
3. specific binding
4. catalysis
5. release of products
Interaction with DNA
Consequently, restriction endonucleases have at least three major
conformations:
apoenzyme (not bound to DNA)
non-specific (non-cognate) DNA binding
specific (cognate) DNA binding
EcoRV
apoenzyme
non-specific
specific
BamHI
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DNA Binding and Target Site Location
Restriction endonucleases bind DNA not only specifically but also, with
considerably weaker affinity, non specifically.
Water molecules (70-80) are released upon non-specific complex formation.
Upon non-specific interaction
the structure of the enzyme
changes:
EcoRV
→ allows interaction with DNA
→ catalytic center far away
from DNA
BamHI
→ no basespecific contacts
apoenzyme
non-specific
complex
BamHI
DNA differs only in one base pair
from the recognition sequence!
→ but non-specific binding
PDBid 1ESG
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DNA Binding and Target Site Location
Non-specific DNA binding is the prerequisite for one-dimensional diffusion
of the protein along the DNA.
One-dimensional diffusion:
Translation of the protein in a non free state along the DNA track.
→ sliding (helical movement along the a groove of the DNA)
→ hopping ( movement parallel to the DNA)
Protein does not leave the DNA
→ intersegment transfer (requires two binding sites on the protein)
DNA Binding and Target Site Location
Sliding is the most important process in target site location.
→ Leaving the target side might also involve sliding etc.
Sliding accelerates target site location:
→ under optimum conditions it allows for scanning of ~106 bases
per binding event.
Really ?
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DNA Binding and Target Site Location
Sliding is the most important process in target site location.
→ Leaving the target side might also involve sliding etc.
Sliding accelerates target site location:
→ under optimum conditions it allows for scanning of ~106 bases
per binding event.
→ but it’s a random walk →the effective sliding distance is much
shorter ~ 1000 bp
DNA Binding and Target Site Location
Sliding is the most important process in target site location.
→ Leaving the target side might also involve sliding etc.
Sliding accelerates target site location:
→ under optimum conditions it allows for scanning of ~106 bases
per binding event.
→ but it’s a random walk →the effective sliding distance is much
shorter ~ 1000 bp
→ ionic conditions, in particular Mg2+ influence sliding distance
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DNA Binding and Target Site Location
Sliding is the most important process in target site location.
→ Leaving the target side might also involve sliding etc.
Sliding accelerates target site location:
→ under optimum conditions it allows for scanning of ~106 bases
per binding event.
→ but it’s a random walk →the effective sliding distance is much
shorter ~ 1000 bp
→ ionic conditions, in particular Mg2+ influence sliding distance
EcoRI follows the helical pitch
→ does not overlook recognition sites
→ pauses at sites that resemble recognition sites
Is it really that easy ?
DNA Binding and Target Site Location
Sliding is the most important process in target site location.
→ Leaving the target side might also involve sliding etc.
Sliding accelerates target site location:
→ under optimum conditions it allows for scanning of ~106 bases
per binding event.
→ but it’s a random walk →the effective sliding distance is much
shorter ~ 1000 bp
→ ionic conditions, in particular Mg2+ influence sliding distance
EcoRI follows the helical pitch
→ does not ovelook reckognition sites
→ pauses at sites that resemble recognition sites
But its stopped by proteins tightly bound to the DNA or unusual DNA
structures
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DNA Recognition
EcoRV system provides an excellent example for the structural changes
occurring upon formation of the specific complex.
apoenzyme
non-specific
complex
specific
complex
All parts of EcoRV are ordered → especially the dimer interfaces and loops
involved in DNA recognition
DNA Recognition
EcoRV
apoenzyme
non-specific
complex
specific
complex
Involves numerous base contacts → allows the enzyme to detect the sequence
12-20 hydrogen bonds are formed
Only few water mediated contacts → increase in contact surface area between
enzyme and DNA (wraps around the DNA)
The B DNA conformation is distorted → here the DNA is bent by ~50°
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DNA Recognition
EcoRV
apoenzyme
non-specific
complex
specific
complex
Enzyme producing 3’ overhangs → mainly use β-stand and β-turn motif
Enzyme producing 5’ overhangs → mainly use an α-helix and a loop
Overview of Co-crystal Structures
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Mechanism of Catalysis
The catalysis of phosphodiester bond cleavage by restriction endonucleases can
be considered as a phosphoryl transfer to water.
Principle mechanisms of phosphoryl transfer reactions:
Suggestions ?
Mechanism of Catalysis
Overall Reaction (EcoRV)
Suggestions ?
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Mechanism of Catalysis
Both mechanisms differ in the amount of bond formation and bond breakage
in the transition state.
associative
dissociative
Alkaline phosphatase seems to achieve substantial catalysis via a dissociative
transition state.
Mechanism of Catalysis
All type II restriction endonucleases, whose crystal structures have been
determined have a catalytic sequence motif in common:
… P D … D/E X K …
where X is any amino acid
For the great majority of typ II restriction
endonucleases Mg2+ is an essential
cofactor.
→ can be substituted with
Mn2+, Fe2+, Co2+, Ni2+, Zn2+
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Mechanism of Catalysis
The understanding of the mechanism of DNA cleavage by Type II restriction
enzymes is critically dependent on the knowledge of how many Mg2+ ions
are involved.
… P D … D/E X K …
The two carboxylates are Mg2+ ligands and the Lysine contacts
the general acid (water molecule).
Mechanism of Catalysis
The second Mg2+ is not involved in catalysis despite the conserved
contact with the phosphate backbone.
Hydrolysis takes place in a single step.
The reaction proceeds with an inversion of the stereochemical configuration
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Mechanism of Catalysis
The nucleophile is a OH- the general base is the substrate itself.
→ substrate assisted catalysis
The 3’ phosphate is the general base, activating a water molecule to attack
the 5’ phosphate.
Mechanism of Catalysis
The general acid is also a water molecule, liganded to both Mg2+ and the
catalytic Lysine.
The Mg2+ ion interacting with the general acid also functions to
stabilize the transition state.
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Comparison of Active Sites
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