Question

What are restriction enzymes, where are the isolated from (and how does this relate to the name of othe restriction enzyme?) what do they do, and how are they used in making recombinant organisms?

Answer 1

Restriction enzymes (RE) are enzymes that cleave the DNA at specific sites. They recognise specific 4-6 unique set of nucleotides in the DNA. The sequences are palindromic, indicating that the complimentary DNA strand has the same sequence of nucleotide. However, these nucleotides are in the reverse direction. Both strands are cut at the same location.

Restriction endonucleases are of three types: type I, II and III. Type II restriction enzymes cleave the DNA within the specific palindromic sequence. However, type I and III restriction enzymes cleave the DNA outside the palindromic sequence. Type II are preferred for recombinant DNA as they can generate cleavages in the specific sequence, and allow insertion of the foreign DNA in t the plasmid.

Restriction enzymes are typically found in bacteria and are required for host defense. They prevent foreign DNA that enters the bacterial cell (for example viruses). The restriction modification system, methylates its own DNA at this specific restriction sites, thereby preventing them from being cleaved by the restriction enzyme. Methylation is the transfer of a methyl group from S-adenosylmethionine to an adenine in the restriction site. The DNA becomes resistant to cleavage by the nuclease that cleaves this same hexanucleotide sequence. Foreign DNA is not cleaved, as it is not methylated.

Naming of RE: The first three letters denotes the bacterial species of origin. The fourth letter denotes the individual strain of bacteria. For example, the restriction system from E. coli

strain K is EcoK. In case there is more than one such enzyme system present in the same bacteria, the different enzymes are given a fifth roman numeral. For example, ECOR1 is present is E. coli strain R, and HindII is one of three enzymes present in d strain of Haemophilus influenzae.

REs can cleave specific sites in the DNA. Hence, they are used in recombinant DNA technology. Recombinant DNA technology is used to produce cloned organisms. The gene of interest is placed in a circular piece of plasmid vector.

The plasmid or vector and the DNA (gene) to be cloned is cleaved by the same RE separately. The plasmid is linearised due the RE cleavage. Type II RE are used as they can generate blunt or sticky ends, depending on where the cut is made. If the cut is made in the center of DNA sequence, a blunt end is formed. If the cut is closer to one end of the sequence, sticky ends are generated. Blunt ends lack overhangs while sticky ends will have overhangs, which complement the sticky ends in the other DNA molecule.

Blunt end:

SmaeI recognises the palindromic sequence CCCGGG and cleaves between C and G

5’CCCGGG3’

3’GGGCCC5’

Ends generated are

5’CCC     GGG3’

3’GGG      CCC5’

Sticky end:

ECOR1 recognises the palindromic sequence GAATTC and cleaves between G and A.

Hence the ends generated are:

5’ G      AATTC3’

3’CTTAA       G 5’

When the vector and the DNA are mixed together, the ends of the vector join with the ends of the DNA to be cloned, to form a recombinant plasmid. Next, the recombinant plasmid is introduced into bacteria cells. Bacteria carrying the plasmid are selected mostly by use of antibiotics and grown. As the bacteria reproduce, the recombinant plasmid is replicated and passed on to the offspring. The bacteria will now make copies of the DNA it contains. Recombinant plants and animals can also be made by this technology.