My watch list
my.chemeurope.com  
Login  

DNA ligase



DNA ligase repairing chromosomal damage.
ligase I, DNA, ATP-dependent
Identifiers
Symbol LIG1
Entrez 3978
HUGO 6598
OMIM 126391
RefSeq NM_000234
UniProt P18858
Other data
EC number 6.5.1.1
Locus Chr. 19 [1]
ligase III, DNA, ATP-dependent
Identifiers
Symbol LIG3
Entrez 3980
HUGO 6600
OMIM 600940
RefSeq NM_002311
UniProt P49916
Other data
Locus Chr. 17 q11.2-q12
ligase IV, DNA, ATP-dependent
Identifiers
Symbol LIG4
Entrez 3981
HUGO 6601
OMIM 601837
RefSeq NM_002312
UniProt P49917
Other data
Locus Chr. 13 q33-q34

In molecular biology, DNA ligase is a particular type of ligase (EC 6.5.1.1) that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA). The alternative, a single-strand break, is easily fixed by DNA polymerase using the complementary strand as a template but still requires DNA ligase to create the final phosphodiester bond to fully repair the DNA.

DNA ligase has applications in both DNA repair and DNA replication (see Mammalian ligases). In addition, DNA ligase has extensive use in molecular biology laboratories for Genetic recombination experiments (see Applications in molecular biology research).

Additional recommended knowledge

Contents

Ligase mechanism

The mechanism of DNA ligase is to form covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another. ATP is required for the ligase reaction.

A pictorial example of how a ligase works (with sticky ends):


Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.

Mammalian ligases

In mammals, there are four specific types of ligase.

  • DNA ligase I: ligates Okazaki fragments during lagging strand DNA replication and some recombinant fragments.
  • DNA ligase II: alternatively spliced form of DNA ligase III found in non-dividing cells.
  • DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing base excision mutations and recombinant fragments.
  • DNA ligase IV: complexes with XRCC4. It catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway. It is also required for V(D)J recombination, the process which generates diversity in immunoglobulin and T-cell receptor loci during immune system development.

Applications in molecular biology research

DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.

One vital, and often tricky, aspect to performing successful recombination experiments involving ligase is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4) which is most active at 25°C. However in order to perform successful ligations, the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated.

If the ambient temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding. The shorter the DNA fragments, the lower the Tm. Thus for sticky ends (overlaps) less than ten base pairs long, ligation experiments are performed at very low temperatures (~4-8°C) for a long period of time (often overnight).

The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli or other bacteria.

See also


 
This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "DNA_ligase". A list of authors is available in Wikipedia.
Your browser is not current. Microsoft Internet Explorer 6.0 does not support some functions on Chemie.DE