What Are Endonucleases?
Various enzymes are involved in tampering with DNA by breaking and forming the bonds between nucleotides. One is a nuclease, a class of enzyme that hydrolyzes the polynucleotide chains of nucleic acid substrates.
Based on where the enzyme makes its cut or the enzyme’s specific cleavage location, the nuclease is sub-categorized into two groups: endonuclease and exonuclease.
- Exonuclease: A group of enzymes that cleave DNA sequences of a nucleic acid chain at the two ends, i.e., at either the 3’ or 5’ termini. Examples are Xrn1 and exonuclease I.
- Endonuclease: A group of enzymes that break the phosphodiester bond present within the polynucleotide chain of a DNA molecule. Its examples include EcoRI and BamHI.
There are also Exo-endonuclease enzymes that possess a hybrid property of both endonuclease and exonuclease enzymes.
These enzymes can either cut within or at the ends of the polypeptide chains.
Unlike exonucleases, endonuclease enzymes don’t require any free ends to carry out the hydrolysis of their substrate. Also, endonucleases can either cut at random locations or require a specific site or a set of nucleotide sequences to perform the cleavage process.
The sequence-specific endonuclease enzymes that cleave DNA at a specific location are known as restriction endonucleases.
This article will introduce you to the types of endonucleases, how they work, their functions, and their applications in lab workflows.
The Endonuclease Mechanism
Non-specific endonucleases degrade RNA or DNA nucleotide chains without any specificity of the sequence. They bind and cleave the DNA molecule at any random site. These enzymes are generally used in labs to clean DNA contamination.
However, restriction endonucleases prefer a specific set of nucleic acid bases or nucleotide sequence to break the phosphodiester bond within a polynucleotide chain.
The nucleotide sequences recognized by the endonuclease to perform the cleavage are called recognition sites. These often consist of 4-6 nucleotides that make a palindromic sequence.
Restriction enzymes identify and bind at the recognition site and break the phosphodiester bonds between the nucleotides. The breakage of these bonds results in either uneven cuts, which form sticky ends (having single-stranded DNA at ends), or blunt ends (no overhangs or single-stranded DNA at 5’ and 3’ termini).
The resulting DNA fragments or oligonucleotides after the cut are joined together by the enzyme DNA ligase, which leads to the formation of recombinant DNA.
Restriction endonucleases are site-specific endonucleases. These enzymes scan the whole length of the DNA molecule, identify and bind to the recognition sequences or cleavage sites, and make a cut at or near those sites.
There is a wide range of restriction enzymes available, each with its own specific cleavage site, which results in different DNA fragments or oligonucleotides. Around 3,600 restriction endonucleases are known, with 250 different sequence specificities.
Image: An illustration of different restriction enzymes, their recognition sequences, and the types of ends produced after their cleavage action.
Restriction enzymes were originally found in archaea and eubacteria, acting as their defense mechanism against viruses (such as phages).
In bacteria, while the host is protected by the methylation of their DNA molecule, restriction enzymes detect foreign DNA and prevent its replication by cutting it into many pieces.
Image: An illustration of a restriction endonucleases in action, utilizing its bacterial defense mechanism against a phage virus.
The restriction enzyme is one of four types based on the cleavage position, subunit composition or structural characterization, cofactor requirements, and cleavage positions. These four types are:
- Type I Enzyme: This enzyme was first identified in two strains of Escherichia coli (or E.coli). It includes multiple subunits that cleave at sites somewhat far from the recognition sequence — at least at a distance of a thousand base pairs away from the cleavage site.
It requires both S-adenosylmethionine and ATP as cofactors to facilitate its dual role of methylation and enzymatic degradation of nucleic acids.
- Type II Enzyme: This enzyme cleaves at sites near or within the recognition sequence. It requires magnesium ions as a cofactor to perform its process and only possesses restriction digestion or endonuclease activity. The digestion results in a 3’ hydroxyl and 5’ phosphate group at the end of the cleavage sites.
It can be composed of a DNA binding and a DNA cleavage domain (such as a Type IIS enzyme), or a DNA cleavage domain with a DNA modification and DNA sequence-specificity domains (such as a Type IIG enzyme).
Type II enzymes are extensively used in molecular biology and biochem labs for routine gene cloning and genome analysis workflows because of their ability to produce distinct fragments and band patterns during electrophoresis.
- Type III Enzyme: This enzyme is a combination of restriction-and-modification enzymes. It cleaves at a site near the recognition sequence. And, it requires two recognition sites in opposite directions within the chain.
- Type IV Enzyme: This enzyme can recognize modified methylated DNA as shown by the McrBC and Mrr systems of E. coli.
Lab assays like amino acid sequencing have also revealed that there is an extensive variety — more than the four types — of restriction enzymes found in organisms at the molecular level.
Endonucleases are involved in a myriad of metabolic functions in different organisms. Some of them are:
- DNA repair: AP endonucleases repair the lesion generated by depurination. The enzyme recognizes the AP site, makes a cut, and prepares the DNA for repair synthesis.
- Non-specific endonucleases are involved in a range of cellular processes, including:
- Digestion of DNA in nucleotide salvage pathways.
- DNA fragmentation during cell apoptosis
- Nucleotide deletion in DNA repair
- mRNA degradation in gene silencing
- mRNA turnover in transcription regulation
- Mutation: Mutations in endonucleases lead to several diseases, including Xeroderma pigmentosa, an autosomal recessive disease caused by mutations in UV-specific endonuclease; and Sickle Cell anemia, in which the recognition site of the restriction endonuclease is eliminated.
- Endonucleases are also involved in UV-irradiated mammalian cells’ repair and recovery.
Endonucleases are predominantly used in biological labs, including molecular biology, biochemistry, and genetic engineering labs, because of their ability to generate distinct nucleic acid fragments.
Based on the experiment purpose, different endonucleases are utilized in the lab workflows along with other substrates that include primers, buffer, sample DNA, EDTA, DNA polymerases, etc.
Some applications of endonucleases are:
- They are used to insert genes into plasmid vectors for protein production and gene cloning.
- They are used in workflows like gel electrophoresis, DNA fingerprinting, or a combination of allele-specific fluorescent probes and restriction assay using real-time PCR to distinguish gene alleles or single nucleotide polymorphisms.
- They are used to digest the genomic DNA of organisms during performing the Southern Blotting technique.
- Some artificial restriction enzymes have also been developed by scientists for gene editing, causing aberrations in human-infecting viruses, and creating targeted mutagenesis.
- They break DNA strands at specific genomic locations, resulting in homology-directed repair (HDR) or nonhomologous end-joining (NEJ) that can be error-prone and cause mutations.
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