Chpt 9: Restriction Endonuclease

Question 1

Type 2 Endonuclease

• Function
• Example
• where are they found?

Answer 1

1) Function
- Cleaves DNA with in a specific base sequence (recognition site)
- Catalyzes hydrolysis of phosphodiester bonds (bond that connects sugar and phosphate groups in nucleotides)
a) produce 5’ PO4 and 3’ OH
b) Mg activates water which creates a nucleophile that attacks phosphorus

2) Found in Arachea and Bacteria

3) Example: EcoR V
- Function-cleaves methylated DNA
- Recognition Site 5’ GATATC 3’

Question 2

Cognate DNA vs Non Cognate DNA

-Def

Answer 2

Cognate DNA- contains recognition site

Noncognate DNA- no recognition site

Question 3

Mechanism of EcoR V

Answer 3

2 mechanisms Hypothesize

• Both Mechanism produce pentacoordinated Transition state
• Mechanisms differ in # of times displacement occurs

Transition State

• Bipyramidal Geometry
  a) nucleophile at one apex
  b) leaving group at other apex
• Inverts stereochemistry **

Mechanism 1: 2 steps

1) Formation of Covalent Intermediate
2) Hydrolysis to final product
- two inversions of stereochemistry
- RETAINS original conformation of Phosphorus

Mechanism 2: Direct Hydrolysis

• Single inversion of stereochemistry
• Inversion of Conformation of Phosphorus

Experimental Details to differentiate between 2 Mechanism?
Stereochemistry around Phosphorus is difficult to determine thus have Experimental Protocol:
1)Phospohorothioate Labeled DNA-> DNA substrate containing sulfur in place of one oxygen
2) Water contain O18
3) Location of O18 relative to sulfur determines Rxn mechanism
Results:
-Mechanism 2 because stereochemistry of water also inverted

Question 4

Experimental Details to differentiate between 2 Mechanism?

Answer 4

Stereochemistry around Phosphorus is difficult to determine thus have Experimental Protocol:

1) Phospohorothioate Labeled DNA-> DNA substrate containing sulfur in place of one oxygen
2) Water contain O18
3) Location of O18 relative to sulfur determines Rxn mechanism

Results:
-Mechanism 2 because stereochemistry of water also inverted

Question 5

All restriction Endonuclease:

• require?
• characteristics?

Answer 5

• require Magnesium or other Divalent Cations

ARE EVOLUTIONARY RELATED

• alignment of amino acid sequence reveals little sequence homologues
• 3D crystal reveals similarities in active site locations especially locations of residues involved in binging Mg2+ and DNA

Question 6

Crystal Structure of EcoR V

Answer 6

Cognate DNA: Mg2+ have NOT been successful due to cleavage of cognate DNA

Question 7

What is Magnesium bound to in Restriction Endonuclease?

Answer 7

Magnesium is bound by:

• Two Aspartate (Asp) residues of EcoR V
• Phosphoryl oxygen atoms of Cognate DNA
• Water

Question 8

Recognition site of DNA

Answer 8

Recognition site proves Two fold Rotational Symmetry

• Inverted Repeats
• Restriction endonuclease functions as a homodimer

Question 9

Homodimer Definition

Answer 9

Dimers related by 2 fold rotational Symmetry

Question 10

Binding of EcoR V to DNA

-Mg2+

Answer 10

Mg2+:

-in the absence of Magnesium, EcoR V binds EQUALLY well to cognate DNA and Noncognate DNA

Question 11

How does EcoR V bind to DNA

• cognate DNA
• type of bonds

Answer 11

Recognition site 5’ GATATC 3’

G:C BP H-bond with EcoR V

• backbone O and H-N of gly (diff gly aa on backbone)
• backbone H-N of Asn

A:T BP H-bond with EcoR V

• Back O and H-N of Asn (same Asn aa)
• R group of O of Thr

Question 12

Cognate DNA vs Noncognate DNA

-recognition site

Answer 12

Cognate DNA recognition site DISTORTS

• Middle 5’ TA 3’ distorts and bring phosphate of DNA cleavage site into proximity of Mg binding site in a active site of EcoR V
• The Mg now binds, completing catalytic apparatus

Noncognate DNA does NOT distort
-Lack of distortion in non cognate DNA does not allow binding of Mg, so catalytic apparatus is not assembled/formed

Question 13

Enzyme Specificity: EcoR V

Answer 13

Enzyme specificity is determined by specificity of enzyme ACTION, rather than specificity of substrate binding
-distortion of cognate DNA while non cognate DNA doesn’t distort so no catalytic apparatus formed

Question 14

Distortion of Cognate DNA

Answer 14

produces addition interactions with EcoR V

• the free energy (binding Energy) of cognate and non cognate DNA are similar
• distortion completes the Mg2+ binding site leading to catalysis

Question 15

Methylation of DNA in cognate DNA

Answer 15

Prevents Distortion

-methylation of 5’ Adenine prevents Hydrogen bonding with Asn thus disruption interaction of DNA with enzyme