DNA ligase

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DNA ligase
DNA Repair.jpg
Artist's conception of DNA ligase repairing chromosomal damage
Identifiers
EC no. 6.5.1.1
CAS no. 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 / QuickGO
Search
PMC articles
PubMed articles
NCBI proteins
ligase I, DNA, ATP-dependent
DNA Ligase.jpg
Identifiers
Symbol LIG1
NCBI gene 3978
HGNC 6598
OMIM 126391
RefSeq NM_000234
UniProt P18858
Other data
Locus Chr. 19
Search for
Structures Swiss-model
Domains InterPro
ligase III, DNA, ATP-dependent
Identifiers
Symbol LIG3
NCBI gene 3980
HGNC 6600
OMIM 600940
RefSeq NM_002311
UniProt P49916
Other data
Locus Chr. 17 q11.2-q12
Search for
Structures Swiss-model
Domains InterPro

DNA ligase is a type of enzyme 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.

Contents

DNA ligase is used in both DNA repair and DNA replication (see Mammalian ligases ). In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments (see Research applications ). Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.

Enzymatic mechanism

The image demonstrates how ligase (yellow oval) catalyzes two DNA fragment strands. The ligase joins the two fragments of DNA to form a longer strand of DNA by "pasting" them together. Ligase.svg
The image demonstrates how ligase (yellow oval) catalyzes two DNA fragment strands. The ligase joins the two fragments of DNA to form a longer strand of DNA by "pasting" them together.

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"). Two ATP molecules are consumed for each phosphodiester bond formed. [ citation needed ]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. Adenylylation (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. [2]
A pictorial example of how a ligase works (with sticky ends) Ligation.svg
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.

Types

E. coli

The E. coli DNA ligase is encoded by the lig gene. DNA ligase in E. coli , as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. [3] It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently.[ citation needed ]

The activity of E. coli DNA ligase can be enhanced by DNA polymerase at the right concentrations. Enhancement only works when the concentrations of the DNA polymerase 1 are much lower than the DNA fragments to be ligated. When the concentrations of Pol I DNA polymerases are higher, it has an adverse effect on E. coli DNA ligase [4]

T4

The DNA ligase from bacteriophage T4 (a bacteriophage that infects Escherichia coli bacteria). The T4 ligase is the most-commonly used in laboratory research. [5] It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than 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. [6] 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.[ citation needed ]

Bacteriophage T4 ligase mutants have increased sensitivity to both UV irradiation [7] [8] and the alkylating agent methyl methanesulfonate [9] indicating that DNA ligase is employed in the repair of the DNA damages caused by these agents.

Mammalian

In mammals, there are four specific types of ligase.

  1. DNA ligase 1: ligates the nascent DNA of the lagging strand after the Ribonuclease H has removed the RNA primer from the Okazaki fragments.
  2. DNA ligase 3: complexes with DNA repair protein XRCC1 to aid in sealing DNA during the process of nucleotide excision repair and recombinant fragments. Of the all known mammalian DNA ligases, only ligase 3 has been found to be present in mitochondria.
  3. DNA ligase 4: 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 from eukaryotes and some microbes uses adenosine triphosphate (ATP) rather than NAD. [3]

Thermostable

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 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.10 This exceptional thermostability permits extremely high hybridization stringency and ligation specificity. [11]

Measurement of activity

There are at least three different units used to measure the activity of DNA ligase: [12]

Research applications

DNA ligases have become indispensable tools 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.

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 bacteriophage T4), which is most active at 37 °C. [13] However, for optimal ligation efficiency with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the melting temperature Tm of the sticky ends being ligated, [14] the 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 overhang, the lower the Tm.

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 °C overnight. The absence of stably annealed ends also means that the ligation efficiency is lowered, requiring a higher ligase concentration to be used. [14]

A novel use of DNA ligase can be seen in the field of nano chemistry, specifically in DNA origami.  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. [15]

History

The first DNA ligase was purified and characterized in 1967 by the Gellert, Lehman, Richardson, and Hurwitz laboratories. [16] 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 E. coli 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 bacteriophage T4, E. coli and other bacteria. [17]

Disorders

Genetic deficiencies in human DNA ligases have been associated with clinical syndromes marked by immunodeficiency, radiation sensitivity, and developmental abnormalities,  [16] 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. [18] A list of prevalent diseases caused by lack of or malfunctioning of DNA ligase is 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. [19]

Ataxia-telangiectasia

Mutations in the ATM gene cause  ataxia–telangiectasia. The ATM gene provides instructions for making a protein that helps control cell division 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). [20]

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. [21]

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. [22] Since excessive cell growth is a hallmark of cancer development, targeted chemotherapy that disrupts the functioning of DNA ligase can impede adjuvant cancer forms. Furthermore, it has been shown that DNA ligases can be broadly divided into two categories, namely, ATP- and NAD+-dependent. Previous research has shown that although NAD+-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+-dependent ligase is present in a eukaryotic organism. The presence solely in non-eukaryotic organisms, unique substrate specificity, and distinctive domain structure of NAD+ dependent compared with ATP-dependent human DNA ligases together make NAD+-dependent ligases ideal targets for the development of new antibacterial drugs. [16]

See also

Related Research Articles

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<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Okazaki fragments</span> Parts of lagging strand in DNA replication

Okazaki fragments are short sequences of DNA nucleotides which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication. They were discovered in the 1960s by the Japanese molecular biologists Reiji and Tsuneko Okazaki, along with the help of some of their colleagues.

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<span class="mw-page-title-main">Rolling circle replication</span> DNA synthesis technique

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<span class="mw-page-title-main">Replisome</span> Molecular complex

The replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthesized. The total result is formation of two new double stranded DNA sequences that are exact copies of the original double stranded DNA sequence.

<span class="mw-page-title-main">DNA shuffling</span>

DNA shuffling, also known as molecular breeding, is an in vitro random recombination method to generate mutant genes for directed evolution and to enable a rapid increase in DNA library size. Three procedures for accomplishing DNA shuffling are molecular breeding which relies on homologous recombination or the similarity of the DNA sequences, restriction enzymes which rely on common restriction sites, and nonhomologous random recombination which requires the use of hairpins. In all of these techniques, the parent genes are fragmented and then recombined.

<span class="mw-page-title-main">DNA clamp</span>

A DNA clamp, also known as a sliding clamp, is a protein complex that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because one of the rate-limiting steps in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotides that the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase.

<span class="mw-page-title-main">Prokaryotic DNA replication</span> DNA Replication in prokaryotes

Prokaryotic DNA Replication is the process by which a prokaryote duplicates its DNA into another copy that is passed on to daughter cells. Although it is often studied in the model organism E. coli, other bacteria show many similarities. Replication is bi-directional and originates at a single origin of replication (OriC). It consists of three steps: Initiation, elongation, and termination.

Fragmentation describes the process of splitting into several pieces or fragments. In cell biology, fragmentation is useful for a cell during both DNA cloning and apoptosis. DNA cloning is important in asexual reproduction or creation of identical DNA molecules, and can be performed spontaneously by the cell or intentionally by laboratory researchers. Apoptosis is the programmed destruction of cells, and the DNA molecules within them, and is a highly regulated process. These two ways in which fragmentation is used in cellular processes describe normal cellular functions and common laboratory procedures performed with cells. However, problems within a cell can sometimes cause fragmentation that results in irregularities such as red blood cell fragmentation and sperm cell DNA fragmentation.

<span class="mw-page-title-main">DNA ligase 1</span> Protein-coding gene in the species Homo sapiens

DNA ligase 1 also DNA ligase I, is an enzyme that in humans is encoded by the LIG1 gene. DNA ligase 1 is the only known eukaryotic DNA ligase involved in both DNA replication and repair, making it the most studied of the ligases.

<span class="mw-page-title-main">T7 DNA polymerase</span> Enzyme

T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, in order to carry out its function. This helps stabilize the binding of the necessary protein to the primer-template to improve processivity by more than 100-fold, which is a feature unique to this enzyme. It is a member of the Family A DNA polymerases, which include E. coli DNA polymerase I and Taq DNA polymerase.

<span class="mw-page-title-main">Ligation (molecular biology)</span> Technique for joining nucleic acid fragments

Ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Eukaryotic cells ligases belong to ATP type, and NAD+ - dependent are found in bacteria (e.g. E. coli).

BLESS, also known as breaks labeling, enrichment on streptavidin and next-generation sequencing, is a method used to detect genome-wide double-strand DNA damage. In contrast to chromatin immunoprecipitation (ChIP)-based methods of identifying DNA double-strand breaks (DSBs) by labeling DNA repair proteins, BLESS utilizes biotinylated DNA linkers to directly label genomic DNA in situ which allows for high-specificity enrichment of samples on streptavidin beads and the subsequent sequencing-based DSB mapping to nucleotide resolution.

References

  1. ↑ Pascal JM, O'Brien PJ, Tomkinson AE, Ellenberger T (November 2004). "Human DNA ligase I completely encircles and partially unwinds nicked DNA". Nature. 432 (7016): 473–8. Bibcode:2004Natur.432..473P. doi:10.1038/nature03082. PMID   15565146. S2CID   3105417.
  2. ↑ Lehman IR (November 1974). "DNA ligase: structure, mechanism, and function". Science. 186 (4166): 790–7. Bibcode:1974Sci...186..790L. doi:10.1126/science.186.4166.790. PMID   4377758. S2CID   86549159.
  3. 1 2 Foster JB, Slonczewski J (2010). Microbiology: An Evolving Science (Second ed.). New York: W. W. Norton & Company. ISBN   978-0-393-93447-2.
  4. ↑ Yang Y, LiCata VJ (February 2018). "Pol I DNA polymerases stimulate DNA end-joining by Escherichia coli DNA ligase". Biochemical and Biophysical Research Communications. 497 (1): 13–18. doi: 10.1016/j.bbrc.2018.01.165 . PMID   29409896.
  5. ↑ "Ligases" (PDF). Enzyme Resources Guide. Promega Corporation. pp. 8–14.
  6. ↑ Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I, Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM (July 2013). "Engineered DNA ligases with improved activities in vitro". Protein Engineering, Design & Selection. 26 (7): 471–8. doi: 10.1093/protein/gzt024 . PMID   23754529.
  7. ↑ Baldy MW (1968). "Repair and recombination in phage T4. II. Genes affecting UV sensitivity". Cold Spring Harbor Symposia on Quantitative Biology. 33: 333–8. doi:10.1101/sqb.1968.033.01.038. PMID   4891973.
  8. ↑ Baldy MW (February 1970). "The UV sensitivity of some early-function temperature-sensitive mutants of phage T4". Virology. 40 (2): 272–87. doi:10.1016/0042-6822(70)90403-4. PMID   4909413.
  9. ↑ Baldy MW, Strom B, Bernstein H (March 1971). "Repair of alkylated bacteriophage T4 deoxyribonucleic acid by a mechanism involving polynucleotide ligase". Journal of Virology. 7 (3): 407–8. doi:10.1128/JVI.7.3.407-408.1971. PMC   356131 . PMID   4927528.
  10. ↑ Tomkinson, Alan E; Sallmyr, Annahita (5 September 2013). "Structure and function of the DNA ligases encoded by the mammalian LIG3 gene". Gene. 531 (2): 150–157. doi:10.1016/j.gene.2013.08.061. PMC   3881560 . PMID   24013086.
  11. ↑ "Ampligase- Thermostable DNA Ligase". www.epibio.com. Archived from the original on 2017-06-19. Retrieved 2017-05-15.
  12. ↑ Russell DW, Sambrook J (2001). "Chapter 1: Plasmids and Their Usefulness in Molecular Cloning". Molecular cloning: a laboratory manual. Vol. 1 (3rd ed.). Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory. pp. 1–159. ISBN   978-0-87969-577-4.
  13. ↑ Baneyx F, Lucotte G (1993). Introduction to Molecular Cloning Techniques. Chichester: John Wiley & Sons. p. 156. ISBN   978-0-471-18849-0.
  14. 1 2 Tabor S (May 2001). "DNA ligases". Current Protocols in Molecular Biology. Chapter 3: Unit 3.14. doi:10.1002/0471142727.mb0314s08. ISBN   978-0-471-14272-0. PMID   18265223. S2CID   23944826.
  15. ↑ Bhanjadeo MM, Nayak AK, Subudhi U (2017). "Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice". Biochemical and Biophysical Research Communications. 485 (2): 492–498. doi:10.1016/j.bbrc.2017.02.024. PMID   28189681.
  16. 1 2 3 Shuman S (June 2009). "DNA ligases: progress and prospects". The Journal of Biological Chemistry. 284 (26): 17365–9. doi: 10.1074/jbc.R900017200 . PMC   2719376 . PMID   19329793.
  17. ↑ Weiss B, Richardson CC (April 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 of the United States of America. 57 (4): 1021–8. Bibcode:1967PNAS...57.1021W. doi: 10.1073/pnas.57.4.1021 . PMC   224649 . PMID   5340583.
  18. ↑ Altmann T, Gennery AR (October 2016). "DNA ligase IV syndrome; a review". Orphanet Journal of Rare Diseases. 11 (1): 137. doi: 10.1186/s13023-016-0520-1 . PMC   5055698 . PMID   27717373.
  19. ↑ "xeroderma pigmentosum". Genetics Home Reference. Retrieved 2017-05-15.
  20. ↑ "ataxia-telangiectasia". Genetics Home Reference. Retrieved 2017-05-15.
  21. ↑ "What Is Fanconi Anemia?". NHLBI, NIH. Retrieved 2017-05-15.
  22. ↑ Tomkinson AE, Howes TR, Wiest NE (June 2013). "DNA ligases as therapeutic targets". Translational Cancer Research. 2 (3). PMC   3819426 . PMID   24224145.
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