III. Short questions

Q1. Write the properties of a type III restriction enzyme.

Ans: Type III restriction enzymes have following

• These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away.
  
  
• They have two different subunits, one subunit (M) is responsible for recognition and modification of DNA sequence and other subunit (R) has nuclease action.
  
  
• Mg⁺² ions, ATP are required as cofactor for DNA cleavage and process of cleavage is stimulated by SAM.
  
  
• Cleave only one strand. Two recognition sites in opposite orientation are necessary to break the DNA duplex.

Q2. Write different application of exonuclease III in molecular cloning experiments.

Ans: Exonuclease III is a deoxyribonuclease enzyme which has 3'→5' exonuclease activity in a double stranded DNA. Different applications of exonuclease III are-

• To generate template for DNA sequencing
  
  •  To generate substrate for DNA labeling experiments
  
  •  DNA-protein interaction assays (to find blockage of exonuclease III activity by protein-DNA binding) etc.