Enzymes, Restriction

Restriction enzymes allow DNA to be cut at specific sites nucleic acid hybridization allows the detection of specific nucleic acid sequences DNA sequencing can be used to easily determine the nucleotide sequence of a DNA molecule. 

Restriction enzymes recognize specific recognition sequences and cut the DNA to leave cohesive ends or blunt ends. The ends of restricted DNA molecules can be joined together by ligation to create new recombinant DNA molecules. 

Restriction enzymes have a three-letter name based on the genus and species name of the bacterium from which they were isolated, together with a roman numeral designed to indicate the identity of the enzyme in cases when the bacterium contains several different restriction enzymes. 

DNA fragments in a restriction digest can be separated by size by electrophoresis in polyacrylamide or agarose gel. Polyacrylamide gel is used to separate smaller DNA molecules whilst agarose gel has larger pore sizes and so can separate larger DNA fragments. 

A map showing the position of cut sites for a variety of restriction enzymes is called the restriction map for that DNA molecule. Restriction maps allow comparison between DNA molecules without the need to determine the nucleotide sequence and are also much used in recombinant DNA experiments. 

Another class of highly repetitive sequences is the Alu family, which consist of about 300 bp that are repeated millions of times throughout the genome. Any Alu sequence is at least 85% homologous in base sequence to any other Alu sequence hence, the family of genes has been highly conserved. Each Alu sequence contains a restriction site that is recognized by the Alul restriction enzyme, from which the name of the family of sequences is derived. 

A variety of enzymes break phosphodiester bonds in nucleic acids deoxyribonucleases (DNases) cleave DNA and ribonucleases (RNases) cleave RNA. DNases usually are specific for single- or double-stranded DNA although some DNases can cleave both. DNases can act as exonucleases in which they remove one nucleotide at a time from either the 3 or 5 end of the strand. Other DNases function as endonucleases and are specific for cleaving between particular pairs of bases. 

Some Restriction Endonucleases and Their Cleavage Sites  

Microorganism Name of Enzyme Target Sequence and Cleavage Sites  

Since a restriction enzyme recognizes a unique sequence, the number of cuts and the number of DNA fragments depends on the size of the molecule. In general, restriction sites consisting of four bases will occur more 

Corwin, Alsoph H. (1976). Ira Remsen. In The Robert A. Welch Foundation Conferences on Chemical Research, Vol. 20, Chap. 4. Easton, PA American Chemical Society. 

Hannaway, Owen (1976). The German Model of Chemical Education in America Ira Remsen at Johns Hopkins (1876-1913). Ambvx 23 145-164. 

WQUam A. (1928-1936). Ira Remsen. In Dictionary of American Biography Base Set. New York American Council of Learned Societies. Reproduced in Biography Resource Center. Detroit Gale Group. Also available from http //www. galenet.com/servlet/BioRC . 

WiUiam A., and Norris, James E. (1932). Ira Remsen. Biographical Memoirs National Academy of Sciences 14 207-257. 

A residue is a single molecular unit within a polymer. Residue is thus another term for monomer. Although the term residue is most often used to refer to a specific amino acid within a polypeptide, it is also used to refer to sugars within a carbohydrate molecule and nucleotides within deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 

---

Ramsden J J and Dreier J 1996 Kinetics of the interaction between DMA and the type 1C restriction enzyme SfyR 124/31 Biochemistry Z5 3746-53... 

The mam use of PCR is to amplify or make hundreds of thousands—even mil lions—of copies of a portion of the polynucleotide sequence m a sample of DNA Sup pose for example we wish to copy a 500 base pair region of a DNA that contains a total of 1 million base pairs We would begin as described m Section 28 14 by cleaving the DNA into smaller fragments using restriction enzymes then use PCR to make copies of the desired fragment... 

The basic steps of gene cloning first involve cutting a precise DNA segment (gene) from a donor source DNA by use of a restriction enzyme (Figure 45.2). At the same time, a small looped... 

Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

Fig. 2. (a) Map of pUC19, a commonly used plasmid vector where the numbers correspond to the positions of the various restriction enzyme cuts and (b) nucleic acid composition of pUC19 from position 393 (5 -end) through position 469 (3 -end) (5,7). 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

Exonucleases. Like the endonucleases they are restriction enzymes which act at the 3 or 5 ends of linear DNA by hydrolysing off the nucleotides. Although they are highly specific for hydrolysing nucleotides at the 3 or 5 ends of linear DNA, the number of nucleotides cleaved are time dependent and usually have to be estimated from the time allocated for cleavage. Commercially available exonucleases are used without further purification. 

Figure 7.8 Sequence-specific recognition sites in the major groove of DNA for three restriction enzymes—Eco RI, Bal I, and Sma I. The DNA sequences that are recognized by these enzymes ate represented by tbe color code defined in Figure 7.7.

Only a rather limited number of base pairs is needed to provide unique and discriminatory recognition sites in the major groove. This is illustrated in Figure 7.8, which gives the color codes for the hexanucleotide recognition sites of three different restriction enzymes—Eco Rl, Bal 1, and Sma 1. It is clear that these patterns are quite different, and each can be uniquely recognized by specific protein-DNA interactions. 

Restriction enzymes (Section 28.14) Enzymes that catalyze the cleavage of DNA at specihc sites. 

Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-depen-dent, and they do not modify DNA by methylation or other means. Most importantly, they cut DNA within or near particular nucleotide sequences that they specifically recognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coU has a restriction enzyme, coRI, that recognizes the hexanucleotide sequence GAATTC ... 

---