Difference between T4 DNA Ligase and E Coli DNA Ligase

DNA ligase is a special type of ligase, an enzyme repairing the single-stranded discontinuities in the double-stranded DNA molecules.

Primarily, DNA ligases are involved in joining the gaps forming in DNA at the time of replication, recombination and DNA repair. The mechanism of DNA ligase is the formation of two covalent phosphodiester bonds between the 3′ hydroxyl ends of one nucleotide with a 5′ phosphate terminal of the other.

ATP is needed for the ligase reaction, which occurs in 3 steps –

  • Adenylation of a residue in the active centre of the enzyme releasing pyrophosphate
  • Transfer of the AMP to the 5′ phosphate donor; pyrophosphate bond is formed
  • A phosphodiester bond is formed between the 5′ phosphate of the donor, and the 3′ hydroxyl of the acceptor

T4 DNA Ligase

  • The T4 DNA Ligase, in vivo, catalyzes the sealing of the single-stranded nicks of the double-stranded molecules of DNA. These are commonly used in the joining of 2 strands of DNA between the 5′-phosphate and the 3′-hydroxyl groups of the neighbouring nucleotides in either blunt-ended or a cohesive ended configuration
  • Enzymes have also been seen in catalyzing the joining of the RNA to either RNA or DNA strand in a duplex molecule. However, does not unite single-stranded nucleic acids
  • Covalently used to join restriction fragments with the E.coli DNA ligase
  • E.coli DNA ligase uses NAD+ while the T4 DNA ligase needs ATP
  • These are able to join the blunt ends of DNA fragments while E.coli DNA ligase joins the cohesive terminals produced by the restriction enzymes
  • The T4 enzyme can rapidly join, producing the full base pairs

E Coli DNA Ligase

  • These are derived from E.coli cells and uses NAD+ as a cofactor
  • These are monomeric enzymes of the MW 74 kDa catalyzing the formation of the phosphodiester bond in the duplex DNA, which contains cohesive terminals.
  • It has a narrow substrate specificity, thus making it a useful tool in a particular application
  • These are not considerably active on the blunt-ended substrates
  • These can be heat-inactivated and use NAD as cofactors
  • They are active at a range of temperatures – 4° C – 37° C

Key Difference between T4 DNA Ligase and E Coli DNA Ligase

The table below depicts the difference between T4 DNA Ligase and E Coli DNA Ligase.

T4 DNA Ligase

E Coli DNA Ligase

What it uses – NAD+ or ATP?

T4 DNA ligase needs ATP

E.coli DNA ligase uses NAD+

What does it join?

Can join the blunt ends of DNA fragments

Joins the cohesive terminals produced by the restriction enzymes

Cofactor requirements

Mg2+, ATP

Mg2+, NAD+

Substrates

ds DNA with cohesive, blunt or complementary ends; nicked ends

ds DNA with cohesive, complementary ends; nicked ends

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