Homing endonuclease
Encyclopedia
The homing endonucleases are a type of restriction enzymes typically encoded by intron
s or intein
s. They act on the cellular DNA
of the cells that synthesize them, in the opposite allele
s of the gene
s that encode them.
, similar to transposon
s, because they facilitate the perpetuation of the genetic elements that encode them.
Homing endonuclease recognition sequences are long enough to occur randomly only with a very low probability (approximately once every ), and are normally found in only one instance per genome
. Generally, the gene encoding the endonuclease (the HEG, "homing endonuclease gene") is located within the recognition sequence which the enzyme cuts, thus interrupting and preventing the cut in the DNA that carries the gene HEG.
Prior to transmission, one allele
carries the gene (HEG+) while the other does not (HEG–), and is therefore susceptible to being cut by the enzyme. Once the enzyme is synthesized, it breaks the chromosome in the HEG- allele, initiating a response from the cellular DNA repair
system. The damage is repaired using recombination
, taking the pattern of the opposite, undamaged DNA allele, HEG+, that contains the gene for the endonuclease. Thus, the gene is copied to the allele that initially did not have it and it is propagated through successive generations. This process is called "homing
".
of the natural source organism, and two lower case letters are derived from the name of the species
of that organism. Finally, a roman numeral distinguishes different enzymes found in a the same organism.
For example, we can mention the enzyme PI-TliII that is the second enzyme encoded by an intein
found in the archaea
Thermococcus litoralis
, and H-DreI, the first synthetic homing endonuclease, created in a laboratory from the enzymes I-DmoI and I-CreI
, taken respectively from Desulfurococcus mobilis y Chlamydomonas reinhardtii
.
Currently there are six known structural families. Their conserved structural motif
s are:
of the homing endonuclease PI-Sce revealed two domains: an endonucleolytic centre resembling the C-terminal domain of Drosophila melanogaster
Hedgehog protein, and a second domain (Homing endonuclease-associated Hint domain) containing the protein-splicing active site
.
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
s or intein
Intein
An intein is a segment of a protein that is able to excise itself and rejoin the remaining portions with a peptide bond. Inteins have also been called "protein introns"....
s. They act on the cellular DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
of the cells that synthesize them, in the opposite allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s of the gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s that encode them.
Origin and mechanism
Although the origin and function of homing endonucleases is still being researched, the most recent hypothesis considers them as selfish genetic elementsSelfish DNA
Selfish DNA refers to those sequences of DNA which, in their purest form, have two distinct properties: the DNA sequence spreads by forming additional copies of itself within the genome; and it makes no specific contribution to the reproductive success of its host organism.This idea was sketched...
, similar to transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
s, because they facilitate the perpetuation of the genetic elements that encode them.
Homing endonuclease recognition sequences are long enough to occur randomly only with a very low probability (approximately once every ), and are normally found in only one instance per genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. Generally, the gene encoding the endonuclease (the HEG, "homing endonuclease gene") is located within the recognition sequence which the enzyme cuts, thus interrupting and preventing the cut in the DNA that carries the gene HEG.
Prior to transmission, one allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
carries the gene (HEG+) while the other does not (HEG–), and is therefore susceptible to being cut by the enzyme. Once the enzyme is synthesized, it breaks the chromosome in the HEG- allele, initiating a response from the cellular DNA repair
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
system. The damage is repaired using recombination
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
, taking the pattern of the opposite, undamaged DNA allele, HEG+, that contains the gene for the endonuclease. Thus, the gene is copied to the allele that initially did not have it and it is propagated through successive generations. This process is called "homing
Homing (biology)
Homing is the inherent ability of an animal to navigate towards an original location through unfamiliar areas. This location may be either a home territory, or a breeding spot.-Uses:...
".
Nomenclature
Homing endonucleases are always indicated with a prefix that identifies their genomic origin, followed by a hyphen: "I-" for homing endonucleases encoded within an intron, "PI-" (for "protein instert") for those encoded within an intein. Some authors have proposed using the prefix "F-" ("freestanding") for viral enzymes and other natural enzymes not encoded by introns nor inteins, and "H-" ("hybrid") for enzymes synthesized in a laboratory. Next, a capital letter is derived from the first letter of the name of the genusGenus
In biology, a genus is a low-level taxonomic rank used in the biological classification of living and fossil organisms, which is an example of definition by genus and differentia...
of the natural source organism, and two lower case letters are derived from the name of the species
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
of that organism. Finally, a roman numeral distinguishes different enzymes found in a the same organism.
For example, we can mention the enzyme PI-TliII that is the second enzyme encoded by an intein
Intein
An intein is a segment of a protein that is able to excise itself and rejoin the remaining portions with a peptide bond. Inteins have also been called "protein introns"....
found in the archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
Thermococcus litoralis
Thermococcus litoralis
Thermococcus litoralis is a species of archaea.-Location:Thermococcus litoralis grows near and around deep-sea smoker vents.-Reproduction:...
, and H-DreI, the first synthetic homing endonuclease, created in a laboratory from the enzymes I-DmoI and I-CreI
I-CreI
I-CreI is a homing endonuclease whose gene was first discovered in the chloroplast genome of Chlamydomonas reinhardtii, a species of unicellular green algae. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first such gene isolated...
, taken respectively from Desulfurococcus mobilis y Chlamydomonas reinhardtii
Chlamydomonas reinhardtii
Chlamydomonas reinhardtii is a single celled green alga about 10 micrometres in diameter that swims with two flagella. They have a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and an "eyespot" that senses light.Although widely distributed...
.
Comparison to other restriction enzymes
Homing endonucleases differ from Type II restriction enzymes in the several respects:- Whereas Type II restriction enzymes bind short, usually symmetric, recognition sequenceRecognition sequenceThe recognition sequence, sometimes also referred to as recognition site, of any DNA-binding protein motif that exhibits binding specificity, refers to the DNA sequence , to which the domain is specific...
s of 4 to 8 bpBase pairIn molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
, homing endonucleases bind very long asymmetric recognition sequences of 12 to 40 bp. - Homing endonucleases are more tolerant of degeneration in the recognition sequence. Minor variations in the recognition sequence decrease the activity of homing endonucleases, but do not abolish it.
- Homing endonucleases share structural motifStructural motifIn a chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a supersecondary structure, which appears also in a variety of other molecules...
s that suggest there are four families, whereas it has not been possible to determine simply recognisable and distinguishable families of Type II restriction enzymes. - Homing endonucleases act as monomerMonomerA monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
s or homodimers, and often require associated proteins to regulate their activity or form ribonucleoprotein complexesRibonucleoproteinRibonucleoprotein is a nucleoprotein that contains RNA, i.e. it is an association that combines ribonucleic acid and protein together. A few known examples include the ribosome, the enzyme telomerase, vault ribonucleoproteins, and small nuclear RNPs , which are implicated in pre-mRNA splicing and...
, wherein RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
is an integral component of the catalytic apparatus. Type II restriction enzymes can also function alone, as monomers or homodimers, or with additional protein subunitProtein subunitIn structural biology, a protein subunit or subunit protein is a single protein molecule that assembles with other protein molecules to form a protein complex: a multimeric or oligomeric protein. Many naturally occurring proteins and enzymes are multimeric...
s, but the accessory subunits differ from those of the homing endonucleases. Thus, they can require restriction, modification, and specificity subunits for their action. - Finally, homing endonucleases have a broader phylogeneticPhylogeneticsIn biology, phylogenetics is the study of evolutionary relatedness among groups of organisms , which is discovered through molecular sequencing data and morphological data matrices...
distribution, occurring in all three biological domainsDomain (biology)In biological taxonomy, a domain is the highest taxonomic rank of organisms, higher than a kingdom. According to the three-domain system of Carl Woese, introduced in 1990, the Tree of Life consists of three domains: Archaea, Bacteria and Eukarya...
—the archaeaArchaeaThe Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
, bacteriaBacteriaBacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
and eukarya. Type II restriction enzymes occur only in archaea, bacteria and certain viruses. Homing endonucleases are also expressed in all three compartments of the eukaryotic cell: nucleiCell nucleusIn cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
, mitochondria and chloroplastChloroplastChloroplasts are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.Chloroplasts are green...
s. Open reading frames encoding homing endonucleases have been found in intronIntronAn intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
s, inteinInteinAn intein is a segment of a protein that is able to excise itself and rejoin the remaining portions with a peptide bond. Inteins have also been called "protein introns"....
s, and in freestanding form between genes, whereas genes encoding Type II restriction enzyme genes have been found only in freestanding form, almost always in close association with genes encoding cognate DNA modifying enzymes. Thus, while the Type II restriction enzymes and homing endonucleases share the function of cleaving double-stranded DNA, they appear to have evolved independently.
Structural families
| border="0"> | A | B | ||
| C | ||||
| Dimer of the I-CreI I-CreI I-CreI is a homing endonuclease whose gene was first discovered in the chloroplast genome of Chlamydomonas reinhardtii, a species of unicellular green algae. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first such gene isolated... homing endonuclease. Alpha helices Alpha helix A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier... are shown in green and beta sheet Beta sheet The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet... s in blue. A: The two small pink spheres in the center of the structure are two metal cations necessary for catalysis. The structure shows the saddle that beta strands create to accommodate the DNA. These strands contain the LAGLIDADG motifs that interact with the DNA minor groove. B & C: DNA atoms are shown as spheres, colored according to chemical element Chemical element A chemical element is a pure chemical substance consisting of one type of atom distinguished by its atomic number, which is the number of protons in its nucleus. Familiar examples of elements include carbon, oxygen, aluminum, iron, copper, gold, mercury, and lead.As of November 2011, 118 elements... . |
||||
Currently there are six known structural families. Their conserved structural motif
Structural motif
In a chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a supersecondary structure, which appears also in a variety of other molecules...
s are:
- LAGLIDADG: Every polypeptide has 1 or 2 LAGLIDADG motifs. The sequence LAGLIDADG is a conserved sequence of amino acidAmino acidAmino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s where each letter is a code that identifies a specific residue. This sequence is directly involved in the DNA cutting process. Those enzymes that have only one motif work as homodimers, creating a saddle that interacts with the major groove of each DNA half-site. The LAGLIDADG motifs contribute amino acid residues to both the protein-protein interface between protein domains or subunits, and to the enzyme's active sites. Enzymes that possess two motifs in a single protein chain act as monomers, creating the saddle in a similar way. The first structures to be determined of homing endonucleases (of PI-SceI and I-CreI, both reported in 1997) were both from the LAGLIDADG structural family., The following year, the first structure of a homing endonuclease (I-CreI) bound to its DNA target site was also reported. - GIY-YIG: These have only one GIY-YIG motif, in the N-terminal region, that interacts with the DNA in the cutting site. The prototypic enzyme of this family is I-TevI which acts as a monomer. Separate structural studies have been reported of the DNA-binding and catalytic domains of I-TevI, the former bound to its DNA target and the latter in the absence of DNA.,
- His-Cys box: These enzymes possess a region of 30 amino acids that includes 5 conserved residues: two histidineHistidineHistidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
s and three cysteins. They co-ordinate the metal cation needed for catalysis. I-PpoI is the best characterized enzyme of this family and acts as a homodimer. Its structure was reported in 1998. - H-N-H: These have a consensus sequenceConsensus sequenceIn molecular biology and bioinformatics, consensus sequence refers to the most common nucleotide or amino acid at a particular position after multiple sequences are aligned. A consensus sequence is a way of representing the results of a multiple sequence alignment, where related sequences are...
of approximately 30 amino acids. It includes two pairs of conserved histidineHistidineHistidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
s and one asparagineAsparagineAsparagine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain's functional group. It is not an essential amino acid...
that create a zinc fingerZinc fingerZinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as interaction modules that bind DNA, RNA, proteins, or small molecules...
domain. I-HmuI is the best characterized enzyme of this family, and acts as a monomer. Its structure was reported in 2004. - PD-(D/E)xK: These enzymes contain a canonical nuclease catalytic domain typically found in type II restriction endonucleases. The best characterized enzyme in this family, I-Ssp6803I, acts as a tetramer. Its structure was reported in 2007.
- Vsr-like: These enzymes were discovered in the Global Ocean Sampling Metagenomic Database and first described in 2009. The term 'Vsr-like' refers to the presence of a C-terminal nuclease domain that displays recognizable homology to bacterial Very Short Patch Repair (Vsr) endonucleases.
Domain architecture
The crystal structureCrystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
of the homing endonuclease PI-Sce revealed two domains: an endonucleolytic centre resembling the C-terminal domain of Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
Hedgehog protein, and a second domain (Homing endonuclease-associated Hint domain) containing the protein-splicing active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
.
External links
- REBASERebase (database)REBASE is a database of information about restriction enzymes and DNA methyltransferases....
, a comprehensive restriction enzyme database from New England BiolabsNew England BiolabsNew England Biolabs produces and supplies reagents for the life science industry. NEB offers 230 recombinant and 30 native restriction enzymes for genomic research, as well as nicking enzymes and DNA methylases. It offers products in the areas related to proteomics, DNA Sequencing, and drug...
with links to related literature..
See also
- List of homing endonuclease cutting sites.
- I-CreI homing endonucleaseI-CreII-CreI is a homing endonuclease whose gene was first discovered in the chloroplast genome of Chlamydomonas reinhardtii, a species of unicellular green algae. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first such gene isolated...
. - MeganucleasesMeganucleasesMeganucleases are endodeoxyribonucleases characterized by a large recognition site ; as a result this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-SceI meganuclease would on average require a genome twenty times the size of the...
. - Restriction enzymeRestriction enzymeA Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
. - IntronIntronAn intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
s and inteinInteinAn intein is a segment of a protein that is able to excise itself and rejoin the remaining portions with a peptide bond. Inteins have also been called "protein introns"....
s. - Intragenomic conflict: Homing endonuclease genes.
- TransposonTransposonTransposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
.