Restriction Enzymes: Procedures for chemical isolation of DNA, such as those developed by Avery, MacLeod, and McCarty, usually lead to random breakage of double-stranded molecules into an average length of about 50,000 base pairs. This length is denoted 50 kb, where kb stands for kilobases (1 kb-1000 base pairs). A length of 50 kb is close to the length of double-stranded DNA present in the bacteriophage that infects E. coli.
- The 50-kb fragments can be made shorter by vigorous shearing forces, such as occur in a kitchen blender, but one of the problems with breaking large DNA molecules into smaller fragments by random shearing is that the fragments containing a particular gene, or part of a gene, will be of different sizes.
- In other words, with random shearing, it is not possible to isolate and identify a particular DNA fragment on the basis of its size and sequence content, because each randomly sheared molecule that contains the desired sequence somewhere within it differs in size from all other molecules that contain the sequence.
- In this section we describe an important enzymatic technique that can be used for cleaving DNA molecules at specific sites.
- This method ensures that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment.
- The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria.
- The enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular, short sequences of bases are present.
- These naturally occurring enzymes serve to protect the bacterial cell by disabling the DNA of bacteriophages that attack it.
- Their discovery earned Werner Arber of Switzerland a Nobel Prize in 1978. Technically, the enzymes are known as type II restriction endonucleases.
- The restriction enzyme BamHI is one example; it recognizes the double-stranded sequence:
5'-GGATCC-3'
3'-CCTAGG-5'
and cleaves each strand between the G-bearing nucleotides shown in bold color.
Figure 2.9 shows how the regions that make up the active site of BamHI contact the recognition site (blue) just prior to cleavage, and the cleavage reaction is indicated in Figure 2.10.
Types of Restriction Enzymes
- Table 2.3 lists nine of the several hundred restriction enzymes that are known.
- Most restriction enzymes are named after the species in which they were found.
- BamHI, for example, was isolated from the bacterium Bacillus amyloliquefaciens strain H, and it is the first (I) restriction enzyme isolated from this organism.
- Because the first three letters in the name of each restriction enzyme stand for the bacterial species of origin, these letters are printed in italics; the rest of the symbols in the name are not italicized.
- Most restriction enzymes recognize only one short base sequence, usually four or six nucleotide pairs.
- The enzyme binds with the DNA at these sites and makes a break in each strand of the DNA molecule, producing 3′-OH and 5′-P groups at each position.
- The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site of the enzyme.
- The examples in Table 2.3 show that some restriction enzymes cleave their restriction site asymmetrically (at different sites in the two DNA strands), but other restriction enzymes cleave symmetrically (at the same site in both strands).
Mechanism of Cleavage
- The former leave sticky ends because each end of the cleaved site has a small, single-stranded overhang that is complementary in base sequence to the other end (Figure 2.10).
- In contrast, enzymes that have symmetrical cleavage sites yield DNA fragments that have blunt ends.
- In virtually all cases, the restriction site of a restriction enzyme reads the same on both strands, provided that the opposite polarity of the strands is taken into account; for example, each strand in the restriction site of BamHI reads 5′-GGATCC-3′ (Figure 2.10).
- A DNA sequence with this type of symmetry is called a palindrome.
Key Characteristics of Restriction Enzymes
Restriction enzymes have the following important characteristics:
- Most restriction enzymes recognize a single restriction site.
- The restriction site is recognized without regard to the source of the DNA.
- Because most restriction enzymes recognize a unique restriction site sequence, the number of cuts in the DNA from a particular organism is determined by the number of restriction sites present.
Conclusion
In conclusion, restriction enzymes play a crucial role in DNA research and genetic engineering. Their ability to cleave DNA molecules at specific sites has revolutionized the way we study and manipulate DNA. Understanding the mechanics of restriction enzymes is fundamental in genetic research and applications such as gene splicing and recombinant DNA technology.
FAQs
- Are restriction enzymes naturally occurring?
- Yes, restriction enzymes are naturally occurring proteins primarily found in bacteria.
- Can restriction enzymes cut DNA from any source?
- Yes, restriction enzymes can cut DNA from any source, irrespective of the organism it originates from.
- What are sticky ends in DNA cleavage?
- Sticky ends are single-stranded overhangs produced after asymmetric cleavage by restriction enzymes, facilitating DNA recombination.
- How are restriction enzymes named?
- Restriction enzymes are named after the bacterial species from which they are isolated.
- What are the practical applications of restriction enzymes?
- Restriction enzymes are extensively used in genetic engineering, DNA fingerprinting, and various biotechnological processes.
The DNA Microarray: An Overview
