Endonuclease
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Endonucleases are enzymes that cleave the
phosphodiester bond In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is ...
within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called
restriction endonucleases A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from
exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the ...
s, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.
Restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
s are endonucleases from
eubacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...
and
archaea Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
that recognize a specific DNA sequence. The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site. Typically, a restriction site will be a palindromic sequence about four to six nucleotides long. Most restriction endonucleases cleave the DNA strand unevenly, leaving complementary single-stranded ends. These ends can reconnect through hybridization and are termed "sticky ends". Once paired, the phosphodiester bonds of the fragments can be joined by
DNA ligase DNA ligase is a specific type of enzyme, a ligase, () 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 organ ...
. There are hundreds of restriction endonucleases known, each attacking a different restriction site. The DNA fragments cleaved by the same endonuclease can be joined regardless of the origin of the DNA. Such DNA is called
recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
; DNA formed by the joining of genes into new combinations. ''Restriction endonucleases'' (
restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
s) are divided into three categories, Type I, Type II, and Type III, according to their mechanism of action. These enzymes are often used in
genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
to make
recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
for introduction into bacterial, plant, or animal cells, as well as in synthetic biology. One of the more famous endonucleases is
Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic e ...
.


Categories

Ultimately, there are three categories of
restriction endonucleases A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
that relatively contribute to the cleavage of specific sequences. The types I and III are large multisubunit complexes that include both the
endonucleases Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
and methylase activities. Type I can cleave at random sites of about 1000 base pairs or more from the recognition sequence and it requires ATP as source of energy. Type II behaves slightly differently and was first isolated by Hamilton Smith in 1970. They are simpler versions of the endonucleases and require no ATP in their degradation processes. Some examples of type II restriction endonucleases include ''Bam''HI, ''Eco''RI, ''Eco''RV, ''Hin''dIII, and ''Hae''III. Type III, however, cleaves the DNA at about 25 base pairs from the recognition sequence and also requires ATP in the process.


Notations

The commonly used notation for restriction endonucleases is of the form "''Vwx''yZ", where "''Vwx''" are, in italics, the first letter of the genus and the first two letters of the species where this restriction endonuclease may be found, for example, ''Escherichia coli'', ''Eco'', and ''Haemophilus influenzae'', ''Hin''. This is followed by the optional, non-italicized symbol "y", which indicates the type or strain identification, for example, ''Eco''R for ''E. coli'' strains bearing the drug resistance transfer factor RTF-1, ''Eco''B for ''E. coli'' strain B, and ''Hin''d for ''H. influenzae'' strain d. Finally, when a particular type or strain has several different restriction endonucleases, these are identified by Roman numerals, thus, the restriction endonucleases from ''H. influenzae'' strain d are named ''Hin''dI, ''Hin''dII, ''Hin''dIII, etc. Another example: "''Hae''II" and "''Hae''III" refer to bacterium ''Haemophilus aegyptius'' (strain not specified), restriction endonucleases number II and number III, respectively. The restriction enzymes used in molecular biology usually recognize short target sequences of about 4 – 8 base pairs. For instance, the ''Eco''RI enzyme recognizes and cleaves the sequence 5' – GAATTC – 3'. Restriction endonucleases come in several types. A restriction endonuclease typically requires a recognition site and a cleavage pattern (typically of nucleotide bases: A, C, G, T). If the recognition site is outside the region of the cleavage pattern, then the restriction endonuclease is referred to as Type I. If the recognition sequence overlaps with the cleavage sequence, then the restriction endonuclease
restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
is Type II.


Further discussion

Restriction endonucleases may be found that cleave standard dsDNA (double-stranded DNA), or ssDNA (single-stranded DNA), or even RNA. This discussion is restricted to dsDNA; however, the discussion can be extended to the following: * Standard dsDNA * Non-standard DNA #
Holliday junction A Holliday junction is a branched nucleic acid structure that contains four double-stranded arms joined. These arms may adopt one of several conformations depending on buffer salt concentrations and the sequence of nucleobases closest to the ju ...
s #
Triple-stranded DNA Triple-stranded DNA (also known as H-DNA or Triplex-DNA) is a DNA structure in which three oligonucleotides wind around each other and form a triple helix. In triple-stranded DNA, the third strand binds to a B-form DNA (via Watson–Crick base ...
, quadruple-stranded DNA (
G-quadruplex In molecular biology, G-quadruplex secondary structures (G4) are formed in nucleic acids by sequences that are rich in guanine. They are helical in shape and contain guanine tetrads that can form from one, two or four strands. The unimolecular ...
) # Double-stranded hybrids of DNA and RNA (one strand is DNA, the other strand is RNA) # Synthetic or artificial DNA (for example, containing bases other than A, C, G, T, refer to the work of
Eric T. Kool Eric T. Kool is an American chemist, focusing in chemistry of RNA and DNA; probe design and imaging; synthetic biology, currently the George A. and Hilda M. Daubert Professor in Chemistry at Stanford University and is an Elected Fellow at the Ameri ...
). Research with synthetic
codons The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
, refer to the research by S. Benner, and enlarging the amino acid set in polypeptides, thus enlarging the proteome or
proteomics Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
, see the research by P. Schultz. In addition, research is now underway to construct synthetic or artificial restriction endonucleases, especially with recognition sites that are unique within a genome. Restriction endonucleases or
restriction enzymes A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
typically cleave in two ways: blunt-ended or sticky-ended patterns. An example of a Type I restriction endonuclease. Furthermore, there exist
DNA/RNA non-specific endonuclease In molecular biology, enzymes in the DNA/RNA non-specific endonuclease family of bacterial and eukaryotic endonucleases share the following characteristics: they act on both DNA and RNA, cleave double-stranded and single-stranded nucleic aci ...
s, such as those that are found in ''
Serratia marcescens ''Serratia marcescens'' () is a species of rod-shaped, Gram-negative bacteria in the family Yersiniaceae. It is a facultative anaerobe and an opportunistic pathogen in humans. It was discovered in 1819 by Bartolomeo Bizio in Padua, Italy.Serrati ...
'', which act on dsDNA, ssDNA, and RNA.


DNA repair

Endonucleases play a role in DNA repair.
AP endonuclease Apurinic/apyrimidinic (AP) endonuclease is an enzyme that is involved in the DNA base excision repair pathway (BER). Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of t ...
, specifically, catalyzes the incision of DNA exclusively at AP sites, and therefore prepares DNA for subsequent excision, repair synthesis and DNA ligation. For example, when depurination occurs, this lesion leaves a deoxyribose sugar with a missing base. The AP endonuclease recognizes this sugar and essentially cuts the DNA at this site and then allows for DNA repair to continue. ''E. coli'' cells contain two AP endonucleases: endonuclease IV (endoIV) and exonuclease III (exoIII) while in eukaryotes, there is only one AP endonuclease.


DNA crosslink repair

Repair of DNA in which the two complementary strands are joined by an interstrand covalent crosslink requires multiple incisions in order to disengage the strands and remove the damage. Incisions are required on both sides of the crosslink and on both strands of the duplex DNA. In mouse embryonic stem cells, an intermediate stage of crosslink repair involves production of double-strand breaks.Hanada K, Budzowska M, Modesti M, Maas A, Wyman C, Essers J, Kanaar R. The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks. EMBO J. 2006 Oct 18;25(20):4921-32. doi: 10.1038/sj.emboj.7601344. Epub 2006 Oct 12. PMID: 17036055; PMCID: PMC1618088
MUS81 Crossover junction endonuclease MUS81 is an enzyme that in humans is encoded by the ''MUS81'' gene. In mammalian somatic cells, MUS81 and another structure specific DNA endonuclease, XPF (ERCC4), play overlapping and essential roles in completion ...
/ EME1 is a structure specific endonuclease involved in converting interstrand crosslinks to double-strand breaks in a DNA replication-dependent manner. After introduction of a double-strand break, further steps are required to complete the repair process. If a crosslink is not properly repaired it can block
DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
.


Thymine dimer repair

Exposure of bacteriophage (phage) T4 to
ultraviolet Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nanometer, nm (with a corresponding frequency around 30 Hertz, PHz) to 400 nm (750 Hertz, THz), shorter than that of visible light, but longer than ...
irradiation induces thymine dimers in the phage DNA. The phage T4 ''denV'' gene encodes endonuclease V that catalyzes the initial steps in the repair of these UV-induced thymine dimers. Endonuclease V first cleaves the glycosylic bond on the 5’ side of a pyrimidine dimer and then catalyzes cleavage of the DNA phospodiester bond that originally linked the two nucleotides of the dimer. Subsequent steps in the repair process involve removal of the dimer remnants and repair synthesis to fill in the resulting single-strand gap using the undamaged strand as template.


Common endonucleases

Below are tables of common prokaryotic and eukaryotic endonucleases.


Mutations

Xeroderma pigmentosa Xeroderma pigmentosum (XP) is a genetic disorder in which there is a decreased ability to repair DNA damage such as that caused by ultraviolet (UV) light. Symptoms may include a severe sunburn after only a few minutes in the sun, freckling in sun- ...
is a rare, autosomal recessive disease caused by a defective UV-specific endonuclease. Patients with mutations are unable to repair DNA damage caused by sunlight. Sickle Cell anemia is a disease caused by a point mutation. The sequence altered by the mutation eliminates the recognition site for the restriction endonuclease MstII that recognizes the nucleotide sequence. tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia. Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders that is caused by mutations in three of the four different subunits of the tRNA-splicing endonuclease complex.


See also

*
Exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the ...
* Restriction endonuclease *
Nuclease A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their ta ...
*
Ribonuclease Ribonuclease (commonly abbreviated RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the ...
*
AP endonuclease Apurinic/apyrimidinic (AP) endonuclease is an enzyme that is involved in the DNA base excision repair pathway (BER). Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of t ...
*
HUH-tag HUH endonucleases (HUH-tags) are sequence-specific single-stranded DNA (ssDNA) binding proteins originating from numerous species of bacteria and viruses. Viral HUH endonucleases are involved in initiating rolling circle replication while ones of ...


References

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