Endonuclease

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Introductions

Endonucleases are a class of enzymes that can recognize and attach specific nucleotide sequences and cleave the phosphodiester bond between two deoxyribonucleotides at specific sites in each strand, referred to as restriction enzymes. According to the structure of restriction enzymes, the required cleavage sites of cofactors and the mode of action, restriction enzymes can be divided into three types, namely Type I, Type II and Type III. Type I restriction endonucleases catalyze both the methylation of host DNA and the hydrolysis of non-methylated DNA, while Type II restriction endonucleases catalyze only the hydrolysis of non-methylated DNA. Type III restriction endonucleases have both modifying and cognitive cleavage roles.

Distribution

Restriction endonucleases are widely distributed, and at least one restriction endonuclease is found in almost all genera and species of bacteria, with dozens in one genus, for example, 22 in the genus Haemophilus (Haemophilus) have been found. Some strains have very low enzyme content, which is difficult to isolate and characterize; however, in some strains, the enzyme content is very high. For example, pMB4 (EcoRI enzyme) and H. aegyptius (Hal Ⅲ enzyme) of E. coli are high enzyme producing strains. It was reported that from 10g of cells of H. aegyptius, the amount of enzyme that could digest l0g of λ phage DNA could be isolated and purified. Bacteria are a major source of restriction endonucleases, especially the very specific class I restriction endonucleases.

Types

Based on the structure of restriction enzymes, the required cut site of cofactor and the mode of action, restriction enzymes can be classified into three types, namely Type I, Type II and Type III.

• Type I restriction endonucleases enzymes

Type I restriction enzymes have both modification and restriction recognition; they also have the ability to recognize specific base sequences on DNA, and usually the cleavage site can be thousands of bases away from the recognition site.

• Type II restriction endonucleases enzymes

Only recognize cleavage site, modification is carried out by other enzymes. The recognized site is mostly a short palindrome sequence; the base sequence cut is usually the recognized sequence. It is a highly useful restriction enzyme for genetic engineering.

• Type III restriction endonucleases enzymes

Similar to type I restriction enzymes, they have both modifying and recognizing functions. It can recognize short asymmetric sequences, and the cleavage site is about 24-26 base pairs away from the recognition sequence.

Applications

• For the assembly of physical maps of DNA genomes, localization of genes and gene isolation, base sequence analysis of DNA molecules, comparing related DNA molecules and genetic engineering, performing genetic engineering editing.
• Restriction endonucleases are produced by bacteria, and their physiological significance is to improve their own defense capabilities.
• Restriction enzymes generally do not cut their own DNA molecules, only exogenous DNA.

Physiological significance

• Restriction is actually a protective mechanism that limits enzymatic degradation of exogenous DNA and maintains genetic stability of the host. Methylation is a common modifying effect that protects by methylation of adenine A and cytosine C.
• The methylation serves the purpose of recognizing its own genetic material and foreign genetic material. Therefore, bacteria that can produce a restriction enzyme for defense against viral infestation may have the sequence recognized by the enzyme in their own genome, only that the recognition sequence or the enzyme cut site is methylated.
• The ability to cleave methylated DNA is an intrinsic and unpredictable property of restriction enzymes, therefore, for efficient cleavage of DNA, both DNA methylation and restriction enzyme sensitivity to that type of methylation must be considered.