Inmolecular biology,endonucleases areenzymes thatcleave thephosphodiester bond within apolynucleotide chain (namelyDNA orRNA). Some, such asdeoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while many, typically calledrestriction endonucleases orrestriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ fromexonucleases, 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.[1] Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.[2]
Restriction enzymes are endonucleases fromeubacteria andarchaea that recognize a specific DNA sequence.[3] The nucleotide sequence recognized for cleavage by a restriction enzyme is called therestriction site. Typically, a restriction site will be apalindromic 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 byDNA ligase. 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 calledrecombinant DNA; DNA formed by the joining of genes into new combinations.[4]Restriction endonucleases (restriction enzymes) are divided into three categories, Type I, Type II, and Type III, according to their mechanism of action. These enzymes are often used ingenetic engineering to makerecombinant DNA for introduction into bacterial, plant, or animal cells, as well as insynthetic biology.[5] One of the more famous endonucleases isCas9.
Ultimately, there are three categories ofrestriction endonucleases that relatively contribute to the cleavage of specific sequences. The types I and III are large multisubunit complexes that include both theendonucleases andmethylase 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 includeBamHI,EcoRI,EcoRV,HindIII, andHaeIII. Type III, however, cleaves the DNA at about 25 base pairs from the recognition sequence and also requires ATP in the process.[4]
The commonly used notation for restriction endonucleases[6] is of the form "VwxyZ", 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, andHaemophilus influenzae,Hin. This is followed by the optional, non-italicized symbol "y", which indicates the type or strain identification, for example,EcoR forE. coli strains bearing the drug resistance transfer factor RTF-1,[6]EcoB forE. coli strain B,[7] andHind forH. influenzae straind.[6] Finally, when a particular type or strain has several different restriction endonucleases, these are identified by Roman numerals, thus, the restriction endonucleases fromH. influenzae strain d are namedHindI,HindII,HindIII, etc. Another example: "HaeII" and "HaeIII" refer to bacteriumHaemophilus aegyptius (strain not specified), restriction endonucleases number II and number III, respectively.[4]: 64–64 The restriction enzymes used in molecular biology usually recognize short target sequences of about 4 – 8 base pairs. For instance, theEcoRI enzyme recognizes and cleaves the sequence 5' – GAATTC – 3'.[8]
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 endonucleaserestriction enzyme is Type II.[citation needed]
Endonucleases play a role in many aspects of biological life. Below are a couple examples of processes where endonucleases play a crucial role.
Endonucleases play a role in DNA repair.AP endonuclease, 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.[9] The AP endonuclease recognizes this sugar and essentially cuts the DNA at this site and then allows for DNA repair to continue.[10]E. coli cells contain two AP endonucleases: endonuclease IV (endoIV) and exonuclease III (exoIII) while in eukaryotes, there is only one AP endonuclease.[11]
Repair of DNA in which the two complementary strands are joined by aninterstrand 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.[12]MUS81/EME1 is a structure specific endonuclease involved in converting interstrand crosslinks to double-strand breaks in a DNA replication-dependent manner.[12] 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 blockDNA replication.[citation needed]
Exposure ofbacteriophage (phage) T4 toultraviolet irradiation inducesthymine dimers in the phage DNA. The phage T4denV gene encodesendonuclease V that catalyzes the initial steps in the repair of these UV-induced thymine dimers.[13] Endonuclease V first cleaves the glycosylic bond on the 5’ side of a pyrimidine dimer and then catalyzes cleavage of the DNA phosphodiester 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.[citation needed]
During apoptosis, Apoptotic endonucleaseDFF40 is activated to initiate controlled cellular disassembly. This disintegration is characterized by the cleavage of genomic DNA into specific fragments. The precise role of endonucleases in this context is to cleave the DNA at specific sites, generating fragments with defined lengths. These fragments are then packaged into apoptotic bodies, ensuring a neat and efficient removal of the dying cell without causing inflammation or damage to neighboring cells.[14]
Flap endonuclease 1 (FEN1) and Dna2 endonuclease are integral toDNA replication on the lagging strand, participating in crucial processes such as primer removal andOkazaki fragment processing. Endonucleases are actively involved in processing these fragments by cleaving the phosphodiester bonds between them. This process is integral to the seamless synthesis and joining of Okazaki fragments, contributing to the overall continuity of the newly replicated DNA strand.[15][16]
Endonucleases, more specificallyendoribonuclease, play a crucial role in RNA processing, a fundamental step in gene expression. This process involves the precise cleavage of precursor RNA molecules, guided by endonucleases, to generate functional RNAs essential for various cellular functions. Endonucleases selectively cleave precursor RNAs at specific sites, defining the boundaries of functional RNA segments during RNA processing. The outcome of RNA processing is the production of functional RNA molecules, such astransfer RNAs (tRNAs) andribosomal RNAs (rRNAs). Endonucleases contribute to the precision of this process, ensuring the formation of mature and functional RNA species.
Endonucleases likeRNase P andtRNase Z (ELAC2), shape precursor tRNAs into mature, functional tRNAs, crucial for accurate translation during protein synthesis.[17] In ribosome biogenesis, endonucleases from theRNase III family, likeDROSHA, play a role in processing precursor rRNAs, contributing to the assembly of functional ribosomes.[18]
DICER andDROSHA also from the RNase III family play a role in the processing pre-miRNA to functional miRNA.[19]
The endonucleaseDNase1L2 also contribute prominently to the removal of DNA during the formation of hair and nails. This process is essential for thematuration of hair and nail structures and is crucial for the transformation of cells into durable andkeratinized structures, ensuring the strength and integrity of hair and nails.[20]
Restriction endonucleases may be found that cleave standard dsDNA (double-stranded DNA), or ssDNA (single-stranded DNA), or even RNA.[citation needed] This discussion is restricted to dsDNA; however, the discussion can be extended to the following:
In addition, research is now underway to construct synthetic or artificial restriction endonucleases, especially with recognition sites that are unique within a genome.[citation needed]
Restriction endonucleases orrestriction enzymes typically cleave in two ways: blunt-ended or sticky-ended patterns. An example of a Type I restriction endonuclease.[4]: 64
Furthermore, there existDNA/RNA non-specific endonucleases, such as those that are found inSerratia marcescens, which act on dsDNA, ssDNA, and RNA.[citation needed]
Below are tables of common prokaryotic and eukaryotic endonucleases.[21]
| Prokaryotic Enzyme | Source | Comments |
|---|---|---|
| RecBCD endonuclease | E. coli | Partially ATP dependent; also an exonuclease; functions in recombination and repair |
| T7 endonuclease (P00641) | phage T7 (gene 3) | Essential for replication; preference for single stranded over double stranded DNA |
| T4 endonuclease II (P07059) | phage T4 (denA) | Splits -TpC- sequence to yield 5'-dCMP- terminated oligonucleotides; chain length of product varies with conditions |
| Bal 31 endonuclease | P. espejiana | Also an exonuclease; nibbles away 3' and 5' ends of duplex DNA. A mixture of at least two nucleases, fast and slow.[22] |
| Endonuclease I (endo I;P25736) | E. coli (endA) | Periplasmic location; average chain length of product is 7; inhibited by tRNA; produces double stranded DNA break; produces nick when complexed with tRNA; endo I mutants grow normally |
| Micrococcal nuclease (P00644) | Staphylococcus | Produces 3'-P termini; requires Ca2+; also acts on RNA; prefers single stranded DNA and AT-rich regions |
| Endonuclease II (endo VI, exo III;P09030) | E. coli (xthA) | Cleavage next to AP site; also a 3'→5' exonuclease; phosphomonoesterase on 3'-P termini |
| Eukaryotic Enzyme | Source | Comments |
| Neurospora endonuclease[23] | Neurospora crassa, mitochondria | Also acts on RNA. |
| S1 nuclease (P24021) | Aspergillus oryzae | Also acts on RNA |
| P1-nuclease (P24289) | Penicillium citrinum | Also acts on RNA |
| Mung bean nuclease I | mung bean sprouts | Also acts on RNA |
| Ustilago nuclease (Dnase I)[24] | Ustilago maydis | Also acts on RNA |
| Dnase I (P00639) | Bovine pancreas | Average chain length of product is 4; produces double strand break in presence of Mn2+ |
| AP endonuclease | Nucleus, mitochondria | Involved in DNA Base Excision Repair pathway |
| Endo R[25] | HeLa cells | Specific for GC sites |
| FLAP1 | Nucleus | Responsible for processingOkazaki fragments during DNA replication |
Xeroderma pigmentosa 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.[26]
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.[27]
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.[28]
