Question

How does a restriction nuclease function? Explain.

Answer 1

Hint: By looking at the words ‘restrictions’ and ‘scissors’, we can understand that these are enzymes that can cleave or make cuts in certain molecules. As a result of cleaving, several fragments are produced that can be further ligated with other fragments to produce a hybrid or recombinant structure. Another term for restriction nucleases is ‘molecular scissors’.

Complete answer:
Restriction nucleases are enzymes that are produced by bacteria and cut the DNA molecule at specific target sequences. The strands of DNA are cleaved into fragments by the restriction enzymes. These enzymes recognise certain sequences on the DNA strand known as restriction sites.

Restriction enzymes can be of two types:
> Restriction endonucleases - This enzyme can cleave the DNA from any particular point within the strand.

> Restriction exonucleases - This enzyme can cleave the strand of DNA at its terminal sequences only.

Restriction endonucleases have more widespread uses than restriction exonucleases. They are majorly used in recombinant DNA technology. The sequences that are recognised by the restriction enzymes are palindrome sequences. Palindromes are those sequences that read the same from the opposite directions in the two strands of DNA.

For instance,
- If one strand reads 5’ GAATTC 3’
- The other strand would read 3’ CTTAAG 5’
- To cleave the strand at a particular sequence, the restriction enzyme catalyses the hydrolysis of the bond between two adjacent nucleotides on the strand. The incisions are made through both sugar-phosphate backbones.

Additional information:
 There are four types of restriction enzymes:
• Type I: require ATP and SAM to function and cleave at sites present away from the recognition sequences.
• Type II: require magnesium to function and cleave at a region that is a short distance from the recognition site.
• Type III: require ATP and SAM, and cleave at a short distance from the target sequence.
• Type IV: this enzyme targets modified DNA, i.e. methylated or hydroxymethylated DNA.

Note: The site that is recognised by the restriction enzyme contains a specific nucleotide sequence. The sequence may also be identified by the number of nucleotides or base pairs (between 4 and 8). The number of nucleotides in the recognition sequence also determines how often it occurs in the entire strand. The recognition sequences are different for each restriction enzyme.