Enzymes used to manipulate DNA
Nucleases
Ligases
Modifying enzymes
Nucleases function
Enzymatically cleave nucleic acids by breaking phosphodiester bonds within DNA/RNA
Nucleases catalysis
Most nucleases require metal ions (e.g.,Mg²⁺, Mn²⁺) for activity
Types of nucleases
Exonucleases and endonucleases
Exonucleases
- Remove nucleotides one at a time from the ends of a DNA or RNA strand.
- Important for DNA replication, repair, and degradation
Endonucleases
- Cleavage of bonds within a nucleotide chain (internally).
- Essential for processes such as restriction digestion and recombination.
Significance of nucleases
Vital for genetic engineering, molecular cloning, and diagnostic procedures.
Exonucleases examples
Bal31- removes nucleotides from both chains of a double stranded DNA
Exonuclease III- degrades DNA from 3’ end but 3’ overhanges of >=4 bases are resistant to the enzyme
Exonuclease III- used to generate ‘nested deletions’ from one end
Endonucleases examples
Single strand specific enzyme- S1 nuclease: only cleaves single strands
Non-specific enzyme- DNase I: cuts both single and double stranded DNA. It cuts non-specifically leaving mononucleotides and short oligonucleotides as end product.
Restriction enzymes cut double stranded DNA by recognizing specific sequences
Modifying enzymes
These enzymes are used to modify DNA molecules by addition or removal of specific groups.
The important enzymes are
(a) Alkaline phosphatase
(b) Polynucleotide kinase
(c) Terminal deoxynucleotidyl transferase
(d) Klenow fragment
Alkaline phosphatase
removes phosphates from the ends of DNA chains
Useful in DNA cloning experiments. DNA ligase cannot join two pieces if they lack a
phosphate
Polynucleotide kinase
transfers gamma phosphate of ATP to the 5 end of DNA chain; a kind of opposite function of alkaline phosphatase
Used in:
-preparing DNA fragments for ligation if they lack phosphates at 5 ends.
-labelling DNA fragments with gamma 32P ATP as a phosphate donor
Terminal deoxy-nucleotidyl transferase
adds nucleotides at the 3`ends of DNA chains. It is a template-independent DNA
polymerase
Used in adding:
- homopolymeric tails in cloning experiments.
- labelled single nucleotides to the 3’ ends of DNA strands to prepare probes
Klenow fragment
Used in
1. Labelling DNA (5-3 polymerase activity)
2. Cloning by converting cohesive ends to blunt ends.
(i) The 3 overhangs are removed by 3-5 exonuclease activity and
(ii) The 5 overhangs are filled by 5-3 polymerase activity
