synonymRestriction endonuclease(restriction endonuclease) generally refers to restriction endonuclease
Restrictive endonuclease (also known as restriction endonuclease) can recognize and attach specific nucleotide sequencesDeoxyribonucleotideBetweenPhosphodiester bondA class of enzymes for cutting, referred to as restriction enzymes.According to the structure of the restriction enzyme,CofactorNeeds ofTangential positionAndAction mode, restriction enzymes can be divided into three types, namely Type I, Type II and Type III.Type I restriction endonuclease can catalyze the hostDNAOfMethylationAnd catalyze the hydrolysis of unmethylated DNA;Type II restriction endonucleases only catalyze the hydrolysis of unmethylated DNA.Type Ⅲ restriction endonuclease has the function of modification and cognitive cleavage at the same time.
Generally, it is composed of the first letter of the genus name and the first two letters of the species name of the microorganism, and the fourth letter represents the strain (strain).For example, the restriction enzyme extracted from Bacillus amylolique faciens H is called Bam H, which is recognized differently in the same strain of bacteriaBase sequenceSeveral enzymes with different specificity can be coded into different numbers, such as Hind ⅡHindⅢ, HpaI, Hpa Ⅱ, MboI, Mbo Ⅱ, etc.
Unit definition: the enzyme amount for digesting 1 μ g of λ DNA in one hour in 0.05mL reaction mixture at the specified pH and 37 ℃ is 1 unit.
Property products do not contain non specific nucleic acidshydrolase(10 units of endonuclease and 1 μ g λ DNA, heat preservation for 16 hoursgel electrophoresisThe stability of the map), which is mainly derived fromprokaryoteSo far, about 4000 restriction enzymes have been isolated from nearly 300 different microorganisms.
Distribution area
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Restrictive endonucleases are widely distributed, and at least one restriction endonuclease is found in almost all genera and species of bacteria, many of which have dozens of species in a genus, such asHaemophilusThere are 22 species of Haemophilus.Some strains have very low enzyme content, which is difficult to isolate and characterize;However, in some strains, the enzyme content is very high, such as EColi's pMB4（EcoRIEnzyme) and Haegyptius（Separation and purificationDigestible 10gLambda bacteriophageDNAThe amount of enzyme.Bacteria are restriction enzymes, especiallySpecificityThe main source of very strong class I restriction endonucleases.
Nature of classification
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EnzymaticFunctional characteristics, size andReaction timeRequiredCofactorRestriction endonucleases can be divided into two categories, namely, class I enzymes and class II enzymes.As early as fromEscherichia coliEcoK and EcoB found in are class I enzymes.Its molecular weight is large;Reaction processMedium removal Mg2+In addition, S is also required-adenosine-Lmethionine、ATP；stayDNA moleculeThere is no specific enzymolysis fragment on, which is the most obvious difference between class I and class II enzymes.Therefore, class I enzymes are used as DNA analysisInstrumental valuenot big.Class II enzymes include EcoR IBamH IHind II, Hind III, etc.Its molecular weight is less than 105 Dalton;Only Mg is needed for reaction2+；The most important thing is that there is specificity in the sequence of the recognized specific baseTangency pointTherefore, after the action of class II enzymes, DNA molecules can produce specific enzymatic fragments, which can be separated and identified by gel electrophoresis.
Restriction enzyme recognitionDNA sequenceInPalindrome sequence。Some enzymes have their cutting sites on one side of palindrome (such as EcoR I, BamH I, Hind, etc.), so they can form sticky ends, while other class II enzymes such asAlu I、BsuR I、Bal I、Hal Ⅲ、HPa I、Sma IThe cutting site is in the middle of palindrome sequence, forming a flat end.The cutting sites of Alu I are as follows:
5'-A G^C T-3'
3'-T C^G A-5'
Among the restriction endonucleases found, the recognition sequence of nearly 100 enzymes has been determined.There are many different sources of enzymes with the samebase calling Sequence, this enzyme is called "heterologousIsoenzyme”Isochizomer, restriction endonuclease;Homolytic enzyme）。It should be noted that although these enzymes have the same recognition order, their cut points are not exactly the same.For example, both Xma I and Sma I recognize six nucleotidesCCCGGG，However, the cut point of Xma I is at CCCCGGG, while that of Ema I is at CCCGGGG. The former cuts DNA molecules to form CCGGViscous endThe latter does not form sticky ends (calledFlat end）。Of course, there are also enzymes with the same recognition order and cut point, such as Hap Ⅱ, Hpa Ⅱ, and Mno I, which all have the same cut point in the recognition order CCGG, and Hal Ⅲ and BsuR I also have the same cut point in the recognition order GGCC.
Restriction endonuclease is produced by bacteria, and its physiological significance is to improve its defense ability
Restriction enzymes generally do not cut their own DNA molecules, only cutExogenous DNA。
name
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The name of restriction enzyme depends on the type of bacteria. Take EcoRI as an example:
Name of restriction enzyme
E
Escherichia
Of
co
coli
(species)
R
RY13
(strain)
I
First find
The order in which they are found
type
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Type I restriction enzyme
It also has modification and recognition cutting（restriction）The role of;In addition, it has the ability to recognize specific base sequences on DNA. Generally, its cleavage site is thousands of bases away from the recognition site.For example:EcoB、EcoK。
Type II restriction enzyme
It can only identify cutting,Modificatory effectBy other enzymes.The positions identified are mostly shortPalindrome sequence（palindrome sequence）；The base sequence cut is usually the recognized sequence.yesgenetic engineeringOn the other hand, it is more practical to limit the types of enzymes.For example:EcoRI、HindⅢ。
Type III restriction enzyme
Similar to type I restriction enzyme, it also has the function of modifying and recognizing cleavage.Recognize short asymmetric sequences, cut bits andIdentification sequenceAbout 24-26Base pair。For example:HinfⅢ。
Physiological significance
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Restrictive effectIn fact, it is to limit enzyme degradationExogenous DNA[1]To maintain the genetic stability of the host.MethylationIt is a common modification, which can makeadenineA andcytosine C is protected by methylation.adoptMethylationTo identify oneselfgenetic materialAnd foreign genetic material.Therefore, bacteria that can produce restriction enzymes against virus infectiongenomeThere may be a sequence recognized by the enzyme in, but the recognition sequence orRestriction siteIs methylated.But it doesn't mean that onceMethylationAll restriction enzymes cannot be cut.Most restriction enzyme pairsDNA methylationTherefore, when the target sequence of the restriction enzyme overlaps with the methylation site, there are three possible effects on digestion, namely, no effect, partial effect, and complete blocking.The ability to cut methylated DNA is an intrinsic and unpredictable characteristic of restriction enzymes. Therefore, in order to cut DNA effectively, DNA methylation and restriction enzymes' ability to cut this type of methylated DNA must be considered at the same timesusceptibility。In addition, most commercially restricted enzymes are now specifically used to cut methylated DNA.
