DNA ligase
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This article needs additional citations for verification. (February 2007) |
| DNA ligase | |||||||||
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 DNA ligase repairing chromosomal damage |
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| Identifiers | |||||||||
| EC number | 6.5.1.1 | ||||||||
| CAS number | 9015-85-4 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / EGO | ||||||||
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| ligase I, DNA, ATP-dependent | |
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| Identifiers | |
| Symbol | LIG1 |
| Entrez | 3978 |
| HUGO | 6598 |
| OMIM | 126391 |
| RefSeq | NM_000234 |
| UniProt | P18858 |
| Other data | |
| Locus | Chr. 19 [1] |
| ligase III, DNA, ATP-dependent | |
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| 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 | |
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| 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 specific type of enzyme, a ligase, (EC 6.5.1.1) that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template,[1] with DNA ligase creating 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). Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.
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Ligase mechanism [edit]
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase reaction, which proceeds in three steps:
- adenylation (addition of AMP) of a residue in the active center of the enzyme, pyrophosphate is released;
- transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond;
- formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.[2]
Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.
Mammalian ligases [edit]
In mammals, there are four specific types of ligase.
- DNA ligase I: ligates the nascent DNA of the lagging strand after the Ribonuclease H has removed the RNA primer from the Okazaki fragments.
- DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing DNA during the process of nucleotide excision repair 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 that generates diversity in immunoglobulin and T-cell receptor loci during immune system development.
DNA ligase in E. coli uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond.[3] DNA ligase from eukaryotes and some other microbes uses adenosine triphosphate (ATP) rather than NAD.[3] Also, a number of other structures present in the DNA ligase are the AMP and lysine, both of which are important in the ligation process since they create an intermediate enzyme.
Applications in molecular biology research [edit]
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 aspect to performing efficient recombination experiments involving the ligation of cohesive-ended fragments is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4), which is most active at 25°C. However, for optimal ligation efficiency with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the sticky ends being ligated.[4] If the ambient temperature exceeds Tm, the homologous pairing of the sticky ends would not be stable because the high temperature disrupts hydrogen bonding. Ligation reaction is most efficient when the sticky ends are already stably annealed, disruption of the annealing ends would therefore results in low ligation efficiency. The shorter the overhang, the lower the Tm, typically a 4-base overhang has a Tm of 12-16°C.
Since blunt-ended DNA fragments have no cohesive ends to anneal, the melting temperature is not a factor to consider within the normal temperature range of the ligation reaction. However, the higher the temperature, the less chance that the ends to be joined will be aligned to allow ligation (molecules move around the solution more at higher temperatures). The limiting factor in blunt end ligation is not the activity of the ligase but rather the number of alignments between DNA fragment ends that occur. The most efficient ligation temperature for blunt-ended DNA would therefore be the temperature at which the greatest number of alignments can occur. Therefore, the majority of blunt-ended ligations are carried out at 14-20°C overnight. The absence of a stably annealed ends also means that the ligation efficiency is lowered, requiring a higher ligase concentration to be used.(T4 DNA ligase is the only commercially-available DNA ligase to anneal blunt ends).[4]
History [edit]
The first DNA ligase was purified and characterized in 1967. The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli and other bacteria.[5]
See also [edit]
References [edit]
- ^ Pascal, John M.; O'Brien, Patrick J.; Tomkinson, Alan E.; Ellenberger, Tom (2004). "Human DNA ligase I completely encircles and partially unwinds nicked DNA". Nature 432 (7016): 473–8. doi:10.1038/nature03082. PMID 15565146.
- ^ Lehnman, I. R. (1974). "DNA Ligase: Structure, Mechanism, and Function". Science 186 (4166): 790–7. doi:10.1126/science.186.4166.790. PMID 4377758.
- ^ a b Slonczewski, Joan, and John Watkins. Foster. Microbiology: An Evolving Science. New York: W.W. Norton &, 2009. Print.
- ^ a b Tabor, Stanley. DNA ligases. Chapter in: Current Protocols in Molecular Biology, Book 1. 2001: Wiley Interscience.[page needed]
- ^ Weiss, B.; Richardson, CC (1967). "Enzymatic Breakage and Joining of Deoxyribonucleic Acid, I. Repair of Single-Strand Breaks in DNA by an Enzyme System from Escherichia coli Infected with T4 Bacteriophage". Proceedings of the National Academy of Sciences 57 (4): 1021–8. doi:10.1073/pnas.57.4.1021. PMC 224649. PMID 5340583.
External links [edit]
- DNA Ligase: PDB molecule of the month
- Davidson College General Information on Ligase
- OpenWetWare DNA Ligation Protocol
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