實(shí)驗(yàn)十一DNA的酶切

1、實(shí)驗(yàn)十一 的酶切1Restriction Endonucleases: An OverviewRestriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. Bacteria initially resist infections by new viruses, and this restriction of viral growt

2、h stemmed from endonucleases within the cells that destroy foreign DNA molecules. Among the first of these restriction enzymes to be purified were EcoRI and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found to cleave DNA at specific sites,

3、generating discrete, gene-size fragments that could be re-joined in the laboratory. Researchers were quick to recognize that restriction enzymes provided them with a remarkable new tool for investigating gene organization, function and expression. As the use of restriction enzymes spread among molec

4、ular biologists in the late 1970s, companies such as New England Biolabs began to search for more. Except for certain viruses, restriction enzymes were found only within prokaryotes. Many thousands of bacteria and archae have now been screened for their presence. Analysis of sequenced prokaryotic ge

5、nomes indicates that they are common-all free-living bacteria and archaea appear to code for them. Restriction enzymes are exceedingly varied; they range in size from the diminutive Pvu II (157 amino acids) to the giant Cje I (1250 amino acids) and beyond. Among over 3,000 activities that have been

6、purified and characterized, more than 250 different sequence-specificities have been discovered. Of these, over 30% were discovered and characterized at New England Biolabs. 2The search for new specificities continues, both biochemically, by the analysis of cell-extracts, and computationally, by the

7、 analysis of sequenced genomes. Although most activities encountered today turn out to be duplicates-isoschizomers-of existing specificities, restriction enzymes with new specificities are found with regularity. Beginning in the early 1980s, New England Biolabs embarked on a program to clone and ove

8、rexpress the genes for restriction enzymes. Cloning improves enzyme purity by separating enzymes from contaminating activities present in the same cells. It also improves enzyme yields and greatly simplifies purification, and it provides the genes for sequencing and analysis, and the proteins for x-

9、ray crystallography. Restriction enzymes protect bacteria from infections by viruses, and it is generally accepted that this is their role in nature. They function as microbial immune systems. When a strain of E.coli lacking a restriction enzyme is infected with a virus, most virus particles can ini

10、tiate a successful infection. When the same strain contains a restriction enzyme, however, the probability of successful infection plummets. The presence of additional enzymes has a multiplicative effect; a cell with four or five independent restriction enzymes could be virtually impregnable. 3Restr

11、iction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases) that protect the cells own DNA from cleavage by the restriction enzyme. Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany, but instead of cleavin

12、g the sequence, they methylate one of the bases in each of the DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it.Together, a restriction enzyme and its cognate modification enzyme(s) form a restriction-modi

13、fication (R-M) system. In some R-M systems the restriction enzyme and the modification enzyme(s) are separate proteins that act independently of each other. In other systems, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modification en

14、zyme. 4Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molec

15、ular level there are many more than three different kinds. Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that th

16、ey are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns. 5Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They pr

17、oduce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for DNA analysis and gene cloning. Rather then forming a single family of related proteins, typeII enzymes are a collection of unrelated proteins of many different sorts. Type I

18、I enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they likely arose independently in the course of evolution rather than diverging from common ancestors. The most common type II enzymes are those like HhaI, Hind III and No

19、t I that cleave DNA within their recognition sequences. Enzymes of this kind are the principle ones available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few, (e.g., BbvC I: CCTCAGC) recognize asymmetric DNA sequences because they bind

20、as heterodimers. Some enzymes recognize continuous sequences (e.g.,EcoRI: GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., Bgl I: GCCNNNNNGGC) in which the half-sites are separated. Cleavage leaves a 3-hydroxyl on one

21、 side of each cut and a 5-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200350 amino acid range. 6The next most common type II enzymes, usually referred to as

22、type IIs are those like Fok I and Alw I that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding, the o

23、ther for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some type IIs enzymes are much more active on DNA molecules that contain multiple recogni

24、tion sites. The third major kind of type II enzyme, more properly referred to as type IV are large, combination restriction-and-modification enzymes, 8501250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recogniti

25、on sequences; those that recognize continuous sequences (e.g., Eco57 I: CTGAAG) cleave on just one side; those that recognize discontinuous sequences (e.g., Bcg I: CGANNNNNNTGC) cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enz

26、ymes are varied but their organization are consistent. They comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit. When these enzymes bind to their substrates, they switc

27、h into either restriction mode to cleave the DNA, or modification mode to methylate it. 7Type III enzymesType III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within

28、 the same DNA molecule to accomplish cleavage; they rarely give complete digests. No laboratory uses have been devised for them, and none are available commercially.8一實(shí)驗(yàn)?zāi)康募氨尘昂怂嵯拗菩詢?nèi)切酶是一類能識別雙鏈中特定堿基順序的核酸水解酶，這些酶都是從原核生物中發(fā)現(xiàn)，它們的功能猶似高等功物的免疫系統(tǒng)， 用于抗擊外來的侵襲。限制性內(nèi)切酶以內(nèi)切方式水解核酸鏈中的磷酸二酯鍵， 產(chǎn)生的片段端為，端為。9限制酶的類型根據(jù)限制酶的識別切割特

29、性， 催化條件及是否具有修飾酶活性可分為、型三大類。類和類限制性內(nèi)切酶，在同一蛋白分子中兼有甲基化作用及依賴ATP的限制性內(nèi)切酶活性。 類限制性內(nèi)切酶結(jié)合于特定識別位點(diǎn)，且沒有特定的切割位點(diǎn)，酶對其識別位點(diǎn)進(jìn)行隨機(jī)切割，很難形成穩(wěn)定的特異性切割末端。類限制性內(nèi)切酶在識別位點(diǎn)上切割，然后從底物上解離下來。故類和類酶在基因工程中基本不用。10型酶型酶就是通常指的限制性內(nèi)切酶. 它們能識別雙鏈的特異順序，并在這個順序內(nèi)進(jìn)行切割，產(chǎn)生特異的片段； 型酶分子量較小，僅需Mg2+作為催化反應(yīng)的輔助因子，識別順序一般為個堿基對的反轉(zhuǎn)重復(fù)順序； 型內(nèi)切酶切割雙鏈產(chǎn)生種不同的切口端突出；端突出和平末端。 正是得益于限制性的內(nèi)切酶的發(fā)現(xiàn)和應(yīng)用， 才使得人們能在體外有目的地對遺傳物質(zhì)進(jìn)行改造，從而極大地推動了分子生物學(xué)的興旺和發(fā)展。11酶切反