View source for DNA ligase

{{Refimprove|date=February 2007}}
{{enzyme
| Name = DNA ligase
| EC_number = 6.5.1.1
| CAS_number = 9015-85-4
| IUBMB_EC_number = 6/5/1/1
| GO_code = 0003910
| image = DNA_Repair.jpg
| width =
| caption = DNA ligase repairing chromosomal damage
}} 
{{infobox protein
| Name = [[LIG1|ligase I, DNA, ATP-dependent]]
| caption =
| image = DNA_Ligase.jpg
| width = 200
| HGNCid = 6598
| Symbol = [[LIG1]]
| AltSymbols =
| EntrezGene = 3978
| OMIM = 126391
| RefSeq = NM_000234
| UniProt = P18858
| PDB =
| ECnumber =
| Chromosome = 19
| Arm =
| Band =
| LocusSupplementaryData =
}}
{{infobox protein
| Name = [[LIG3|ligase III, DNA, ATP-dependent]]
| caption =
| image =
| width =
| HGNCid = 6600
| Symbol = [[LIG3]]
| AltSymbols =
| EntrezGene = 3980
| OMIM = 600940
| RefSeq = NM_002311
| UniProt = P49916
| PDB =
| ECnumber =
| Chromosome = 17
| Arm = q
| Band = 11.2
| LocusSupplementaryData = -q12
}}
'''DNA ligase''' is a specific type of enzyme, a [[ligase]], ({{EC number|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 [[LIG4|DNA ligase IV]]) may specifically repair double-strand breaks (i.e. a break in both [[Complementarity (molecular biology)|complementary]] strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template,<ref name="pmid15565146">{{cite journal | vauthors = Pascal JM, O'Brien PJ, Tomkinson AE, Ellenberger T | title = Human DNA ligase I completely encircles and partially unwinds nicked DNA | journal = Nature | volume = 432 | issue = 7016 | pages = 473–8 | date = November 2004 | pmid = 15565146 | doi = 10.1038/nature03082 }}</ref> with DNA ligase creating the final phosphodiester bond to fully repair the DNA.

DNA ligase is used in both [[DNA repair]] and [[DNA replication]] (see ''[[DNA ligase#Mammalian ligases|Mammalian ligases]]'').  In addition, DNA ligase has extensive use in [[molecular biology]] laboratories for [[recombinant DNA]] experiments (see ''[[DNA ligase#Applications in molecular biology research|Applications in molecular biology research]]''). Purified DNA ligase is used in gene cloning to join DNA molecules together to form [[recombinant DNA]].

==Ligase mechanism==
The mechanism of DNA ligase is to form two [[covalent]] [[phosphodiester bonds]] between [[3' end|3' hydroxyl ends]] of one [[nucleotide]] ("acceptor"), with the [[5' end|5' phosphate end]] of another ("donor"). Two ATP molecules are consumed for each phosphodiester bond formed. AMP is required for the ligase reaction, which proceeds in four steps:

1. Reorganization of activity site such as nicks in DNA segments or Okazaki fragments etc.

2. Adenylation (addition of AMP) of a [[lysine]] residue in the active center of the enzyme, [[pyrophosphate]] is released;

3. Transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond; 
4. Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.<ref name="pmid4377758">{{cite journal | vauthors = Lehman IR | title = DNA ligase: structure, mechanism, and function | journal = Science | volume = 186 | issue = 4166 | pages = 790–7 | date = November 1974 | pmid = 4377758 | doi = 10.1126/science.186.4166.790 }}</ref> [[Image:Ligation.svg|thumb|none|upright=2|A pictorial example of how a ligase works (with [[DNA end|sticky end]]s)]]

Ligase will also work with [[DNA end|blunt end]]s, although higher enzyme concentrations and different reaction conditions are required.

== Types of ligases ==

=== ''E. coli'' DNA ligase ===
The ''[[Escherichia coli|E. coli]]'' DNA ligase is encoded by the ''lig'' gene.  DNA ligase in ''[[Escherichia coli|E. coli]]'', as well as most prokaryotes, uses energy gained by cleaving [[nicotinamide adenine dinucleotide]] (NAD) to create the phosphodiester bond.<ref name="foster"/>  It does not ligate blunt-ended DNA except under conditions of molecular crowding with [[polyethylene glycol]], and cannot join RNA to DNA efficiently.

=== T4 DNA ligase ===
The DNA ligase from [[bacteriophage T4]] is the ligase most-commonly used in laboratory research.<ref name="url_www.promega.com">{{cite web | url = https://www.promega.com/~/media/files/resources/product%20guides/cloning%20enzymes/ligases.pdf?la=en+ | title = Ligases | work = Enzyme Resources Guide | publisher = Promega Corporation | pages = 8–14 }}</ref>  It can ligate [[cohesive end|cohesive or "sticky" ends]] of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate [[blunt end|blunt-ended DNA]] with much greater efficiency than ''[[Escherichia coli|E. coli]]'' DNA ligase.  Unlike ''E. coli'' DNA ligase, T4 DNA ligase cannot utilize NAD and it has an absolute requirement for ATP as a cofactor. Some engineering has been done to improve the ''in vitro'' activity of T4 DNA ligase; one successful approach, for example, tested T4 DNA ligase fused to several alternative DNA binding proteins and found that the constructs with either p50 or [[NF-kB]] as fusion partners were over 160% more active in blunt-end ligations for cloning purposes than wild type T4 DNA ligase.<ref name="pmid23754529">{{cite journal | vauthors = Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I, Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM | title = Engineered DNA ligases with improved activities in vitro | journal = Protein Engineering, Design & Selection | volume = 26 | issue = 7 | pages = 471–8 | date = July 2013 | pmid = 23754529 | doi = 10.1093/protein/gzt024 }}</ref>  A typical reaction for inserting a fragment into a plasmid vector would use about 0.01 (sticky ends) to 1 (blunt ends) units of ligase.  The optimal incubation temperature for T4 DNA ligase is 16 °C.&nbsp;

=== Mammalian ligases ===
In mammals, there are four specific types of ligase.
#[[LIG1|DNA ligase I]]: ligates the nascent DNA of the [[DNA replication#Lagging strand|lagging strand]] after the [[RNase H|Ribonuclease H]] has removed the RNA primer from the [[Okazaki fragment]]s.
#[[LIG3|DNA ligase III]]: [[protein complex|complexes]] with [[DNA repair]] [[protein]] [[XRCC1]] to aid in sealing DNA during the process of [[DNA repair|nucleotide excision repair]] and recombinant fragments. Of the all known mammalian DNA ligases, only Lig III has been found to be present in mitochondria.
#[[LIG4|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 II: appears to be used in repair. It is formed by alternative splicing of a proteolytic fragment of DNA ligase III and does not have its own gene, therefore it is often considered to be virtually identical to DNA ligase III.

DNA ligase from [[eukaryotes]] and some microbes uses [[adenosine triphosphate]] (ATP) rather than NAD.<ref name="foster">{{cite book | vauthors = Foster JB, Slonczewski J | title = Microbiology: An Evolving Science | edition = Second | publisher = W. W. Norton & Company | location = New York | year = 2010 | pages = | isbn = 978-0-393-93447-2 }}</ref>

===Thermostable ligases===
Derived from a thermophilic bacterium, the enzyme is stable and active at much higher temperatures than conventional DNA ligases. Its half-life is 48 hours at 65°C and greater than 1&nbsp;hour at 95°C. Ampligase DNA Ligase has been shown to be active for at least 500 thermal cycles (94°C/80°C) or 16 hours of cycling.<sup>10</sup>&nbsp;This exceptional thermostability permits extremely high hybridization stringency and ligation specificity.<ref>{{cite web|url=http://www.epibio.com/enzymes/ligases-kinases-phosphatases/dna-ligases/ampligase-thermostable-dna-ligase?details|title=Ampligase- Thermostable DNA Ligase|website=www.epibio.com|access-date=2017-05-15}}</ref>

==Measurement of ligase activity==
There are at least three different units used to measure the activity of DNA ligase:<ref name="Russell_2001">{{cite book | vauthors = Russell DW, Sambrook J | title = Molecular cloning: a laboratory manual | publisher = Cold Spring Harbor Laboratory | location = Cold Spring Harbor, N.Y | year = 2001 | isbn = 978-0-87969-577-4 | volume = 1 | edition = 3rd | chapter = Chapter 1: Plasmids and Their Usefulness in Molecular Cloning  | pages=1–159 }}</ref>
* '''Weiss unit''' - the amount of ligase that catalyzes the exchange of 1 nmole of <sup>32</sup>P from inorganic [[pyrophosphate]] to ATP in 20 minutes at 37<sup>°</sup>C.  This is the one most commonly used.
* '''Modrich-Lehman unit''' - this is rarely used, and one unit is defined as the amount of enzyme required to convert 100 nmoles of d(A-T)<sub>n</sub> to an exonuclease-III resistant form in 30 minutes under standard conditions.
* Many commercial suppliers of ligases use an arbitrary unit based on the ability of ligase to ligate cohesive ends.  These units are often more subjective than quantitative and lack precision.

== Applications in molecular biology research ==
{{main|Ligation (molecular biology) }}
DNA ligases have become indispensable tools in modern molecular biology research for generating [[recombinant DNA]] sequences.  For example, DNA ligases are used with [[restriction enzyme]]s to insert DNA fragments, often [[genes]], into [[plasmid]]s.

Controlling the optimal temperature is a vital aspect of performing efficient recombination experiments involving the ligation of cohesive-ended fragments.  Most experiments use T4 DNA Ligase (isolated from [[Enterobacteria phage T4|bacteriophage T4]]), which is most active at 37&nbsp;°C.<ref name="Baneyx_1993">{{cite book | vauthors = Baneyx F, Lucotte G | title = Introduction to Molecular Cloning Techniques | publisher = John Wiley & Sons | location = Chichester | year = 1993 | page = 156 | isbn = 978-0-471-18849-0 }}</ref>  However, for optimal ligation efficiency with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the [[DNA melting|melting temperature]] T<sub>m</sub> of the sticky ends being ligated,<ref name="Tabor_2001">{{cite journal | vauthors = Tabor S | title = DNA ligases | journal = Current Protocols in Molecular Biology | volume = Chapter 3 | issue =  | pages = Unit 3.14 | date = May 2001 | pmid = 18265223 | doi = 10.1002/0471142727.mb0314s08 | isbn = 978-0-471-14272-0 }}</ref> the [[Homologous chromosome|homologous pairing]] of the sticky ends will not be stable because the high temperature disrupts [[hydrogen bonding]]. A ligation reaction is most efficient when the sticky ends are already stably annealed, and disruption of the annealing ends would therefore result in low ligation efficiency. The shorter the [[DNA end#Overhangs and sticky ends|overhang]], the lower the T<sub>m</sub>.

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. 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.  The majority of blunt-ended ligations are carried out at 14-25&nbsp;°C overnight.  The absence of stably annealed ends also means that the ligation efficiency is lowered, requiring a higher ligase concentration to be used.<ref name="Tabor_2001"/>

A novel use of DNA ligase can be seen in the field of nano chemistry, specifically in DNA origami. &nbsp;DNA based self-assembly principles have proven useful for organizing nanoscale objects, such as biomolecules, nanomachines, nanoelectronic and photonic component. Assembly of such nano structure requires the creation of an intricate mesh of DNA molecules. Although DNA self-assembly is possible without any outside help using different substrates such as provision of catatonic surface of Aluminium foil, DNA ligase can provide the enzymatic assistance that is required to make DNA lattice structure from DNA over hangs.<ref name="pmid28189681">{{cite journal | vauthors = Bhanjadeo MM, Nayak AK, Subudhi U | title = Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice | journal = Biochemical and Biophysical Research Communications | volume = 485 | issue = 2 | pages = 492–498 | year = 2017 | pmid = 28189681 | doi = 10.1016/j.bbrc.2017.02.024 }}</ref>

== History ==
The first DNA ligase was purified and characterized in 1967 by the Gellert, Lehman, Richardson, and Hurwitz laboratories.<ref name="Shuman 17365–17369">{{cite journal | vauthors = Shuman S | title = DNA ligases: progress and prospects | journal = The Journal of Biological Chemistry | volume = 284 | issue = 26 | pages = 17365–9 | date = June 2009 | pmid = 19329793 | pmc = 2719376 | doi = 10.1074/jbc.R900017200 }}</ref> It was first purified and characterized by Weiss and Richardson using a six-step chromatographic-fractionation process beginning with elimination of cell debris and addition of streptomycin, followed by several Diethylaminoethyl (DEAE)-cellulose column washes and a final phosphocellulose fractionation. The final extract contained 10% of the activity initially recorded in the&nbsp;''E. coli&nbsp;''media; along the process it was discovered that ATP and Mg++ were necessary to optimize the reaction. The common commercially available DNA ligases were originally discovered in [[Enterobacteria phage T4|bacteriophage T4]], ''[[Escherichia coli|E. coli]]'' and other [[bacteria]].<ref name="WeissRichardson1967">{{cite journal | vauthors = Weiss B, Richardson CC | title = 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 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 57 | issue = 4 | pages = 1021–8 | date = April 1967 | pmid = 5340583 | pmc = 224649 | doi = 10.1073/pnas.57.4.1021 }}</ref>

== Disorders ==
Genetic deficiencies in human DNA ligases have been associated with clinical syndromes marked by immunodeficiency, radiation sensitivity, and developmental abnormalities,&nbsp;<ref name="Shuman 17365–17369"/> [[LIG4 syndrome]] (Ligase IV syndrome) is a rare disease associated with mutations in DNA ligase 4 and interferes with dsDNA break-repair mechanisms. Ligase IV syndrome causes immunodeficiency in individuals and is commonly associated with microcephaly and marrow hypoplasia.<ref>{{cite journal | vauthors = Altmann T, Gennery AR | title = DNA ligase IV syndrome; a review | journal = Orphanet Journal of Rare Diseases | volume = 11 | issue = 1 | pages = 137 | date = October 2016 | pmid = 27717373 | pmc = 5055698 | doi = 10.1186/s13023-016-0520-1 }}</ref> A list of prevalent diseases caused due to lack of or malfunctioning of DNA ligase are as follows.

=== Xeroderma pigmentosum ===

Xeroderma pigmentosum, which is commonly known as XP, is an inherited condition characterized by an extreme sensitivity to ultraviolet (UV) rays from sunlight. This condition mostly affects the eyes and areas of skin exposed to the sun. Some affected individuals also have problems involving the nervous system.<ref>{{Cite web|url=https://ghr.nlm.nih.gov/condition/xeroderma-pigmentosum|title=xeroderma pigmentosum| work = Genetics Home Reference | access-date = 2017-05-15 }}</ref>

=== Ataxia-telangiectasia ===

Mutations in the&nbsp;ATM&nbsp;gene cause&nbsp;ataxia-telangiectasia. The&nbsp;ATM&nbsp;gene provides instructions for making a protein that helps control&nbsp;cell&nbsp;division&nbsp;and is involved in DNA repair. This protein plays an important role in the normal development and activity of several body systems, including the nervous system and immune system. The ATM protein assists cells in recognizing damaged or broken DNA strands and coordinates DNA repair by activating enzymes that fix the broken strands. Efficient repair of damaged DNA strands helps maintain the stability of the cell's genetic information. Affected children typically develop difficulty walking, problems with balance and hand coordination, involuntary jerking movements (chorea), muscle twitches (myoclonus), and disturbances in nerve function (neuropathy). The movement problems typically cause people to require wheelchair assistance by adolescence. People with this disorder also have slurred speech and trouble moving their eyes to look side-to-side (oculomotor apraxia).<ref>{{cite web | url = https://ghr.nlm.nih.gov/condition/ataxia-telangiectasia#genes | title = ataxia-telangiectasia | work = Genetics Home Reference | access-date = 2017-05-15 }}</ref>

=== Fanconi Anemia ===

[[Fanconi anemia]] (FA) is a rare, inherited blood disorder that leads to bone marrow failure. FA prevents bone marrow from making enough new blood cells for the body to work normally. FA also can cause the bone marrow to make many faulty blood cells. This can lead to serious health problems, such as [[leukemia]].<ref>{{cite web | url = https://www.nhlbi.nih.gov/health/health-topics/topics/fanconi | title = What Is Fanconi Anemia? | publisher = NHLBI, NIH | access-date = 2017-05-15 }}</ref>

=== Bloom syndrome ===

[[Bloom syndrome]] results in skin that is sensitive to sun exposure, and usually the development of a butterfly-shaped patch of reddened skin across the nose and cheeks. A skin rash can also appear on other areas that are typically exposed to the sun, such as the back of the hands and the forearms. Small clusters of enlarged blood vessels (telangiectases) often appear in the rash; telangiectases can also occur in the eyes. Other skin features include patches of skin that are lighter or darker than the surrounding areas (hypopigmentation or hyperpigmentation respectively). These patches appear on areas of the skin that are not exposed to the sun, and their development is not related to the rashes.

== As a drug target ==
In recent studies, human DNA ligase I was used in [[Computer-aided drug design]] to identify DNA ligase inhibitors as possible therapeutic agents to treat cancer.<ref>{{cite journal | vauthors = Tomkinson AE, Howes TR, Wiest NE | title = DNA ligases as therapeutic targets | journal = Translational Cancer Research | volume = 2 | issue = 3 | date = June 2013 | pmid = 24224145 | pmc = 3819426 }}</ref> Since excessive cell growth is a hallmark of cancer development, targetes chemotherapy that disrupts the functioning of DNA ligase can impede adjuvant cancer forms. Furthermore, it has been shown that DNA ligase can be broadly divided into two categories namely, ATP dependent and NAD+ dependent. Previous research has shown that although NAD<sup>+</sup>-dependent DNA ligases have been discovered in sporadic cellular or viral niches outside the bacterial domain of life, there is no instance in which a NAD<sup>+</sup>-dependent ligase is present in a eukaryal organism. The narrow phylogenetic distribution, unique substrate specificity, and distinctive domain structure of NAD+ depandant compared with ATP-dependent human DNA ligases recommend the NAD<sup>+</sup>&nbsp;ligases as targets for the development of new antibacterial drugs.<ref name="Shuman 17365–17369" />

== See also ==
* [[DNA end]]
* [[Lagging strand]]
* [[DNA replication]]
* [[Okazaki fragment]]
* [[DNA polymerase]]

== References ==
{{Reflist|33em}}

== External links ==
*[http://nist.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb55_1.html DNA Ligase: PDB molecule of the month]
*[http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Weber/ligation.html Davidson College General Information on Ligase]
*[http://openwetware.org/wiki/DNA_ligation OpenWetWare DNA Ligation Protocol]

{{DNA replication}}
{{Phosphoric ester and nitrogen-metal ligases}}
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[[Category:EC 6.5]]
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