PART III Techniques in Molecular Biology CHAPTER 11 RESTRICTION ENDONUCLEASES SHIV SHANKAR1*, IMRAN UDDIN2, and SEYEDEH FATEMEH AFZALI3 1 Department of Food Engineering and Bionanocomposite Research Institute, Mokpo National University, 61 Dorimri, Chungkyemyon, Muangun 534729, Jeonnam, Republic of Korea 2 Nanotechnology Innovation Centre, Department of Chemistry, Rhodes University, PO Box 94, Grahamstown, South Africa 3 Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Malaysia Corresponding author. E-mail: shivbiotech@gmail.com * CONTENTS Abstract .2 Background of Restriction Endonuclease .3 Recognition Sites of Restriction Endonuclease .4 Discovery of Restriction Enzymes or Restriction Endonucleases .5 Types of Restriction Endonucleases .7 Mechanism of Action of Restriction Endonuclease .8 Interaction of Restriction Endonuclease with the DNA.9 Isoschizomers and Neoschizomers .10 Commonly Used Restriction Endonucleases .249 236 Plant Biotechnology: Volume 1 11.11 Recent Development of Restriction Endonucleases .12 Fast Digest Restriction Endonucleases .14 Conclusions and Future Prospect .256 Restriction Endonucleases 237 ABSTRACT Restriction endonucleases are an integral part of genetic engineering. The birth of genetic engineering and the advancement in the molecular tech- niques in modern research were possible due to the discovery of the restric- tion endonucleases. Various types of restriction endonucleases have been discovered and named according to the recognition and cleavage position sites in the DNA sequences.
This chapter has focused on types of restriction endonuclease, their mechanism of action, and the interaction with DNA. At the end of this chapter, recent developments of restriction endonucleases and restriction mapping have been discussed 11.1 GENERAL INTRODUCTION The study of genetic materials (genetic engineering) has contributed signifi- cant advancement in many areas of modern research and development. The birth of genetic engineering was possible due to the discovery of special enzymes that cut DNA. Many endeavors of molecular-level engineering rely on biological material such as nucleic acids and restriction enzymes.
The field of recombinant DNA and genetic engineering depend on enzymes and techniques that permit the precise cutting, splicing, and sequencing DNA molecules; recognition of recombinant products; and the introduction of recombinant molecules into the cells of any organism. The study of gene themselves became possible with the advent of endonuclease enzymes in bacteria. Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some endonucleases, such as deoxyribonu- clease I cut the DNA relatively nonspecifically (without regard to sequence), while many others, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences (Cox et al.
Restriction enzymes are endonucleases that are found in eubacteria and archaea and recognize a specific DNA sequence (Stephen et al. The nucleotide sequence recognized by the restriction enzymes for cleavage is called the restriction site. Generally, the restriction site are a palin- dromic sequence of about four to six nucleotides in length. Most restric- tion endonucleases cut the DNA strand unevenly, leaving complementary single-stranded ends.
These ends can reconnect through hybridization and are called as “sticky ends,” which can be joined through the phosphodiester bonds by the DNA ligase. The hundreds of restriction endonucleases are well-known that are specific for unique restriction sites. The DNA fragments 238 Plant Biotechnology: Volume 1 from different origin that are cut by the same endonuclease can be joined to make recombinant DNA. Recombinant DNA is formed by the joining of two or more genes into new combinations (Cox et al.
Restriction enzymes are usually classified into three types that are different in structure and whether they cut DNA at their recognition site or if their cleavage and recognition sites are separate from one another. To cleave DNA, all restric- tion enzymes make at least two incisions through each sugar–phosphate backbone of the DNA double helix. Restriction enzymes are found in archaea and bacteria that provide a defense mechanism against invading viruses (Albert and Linn, 1969; Kruger and Bickle, 1983). The restriction enzymes selectively cut foreign DNA inside a prokaryote in a process called restriction.
However, the DNA of host organism is protected by a modification by an enzyme, methyltransferase blocks cleavage. These two processes establish the restriction modification system (Kobayashi, 2001). More than 4000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially (Roberts et al. These enzymes have been used routinely for DNA modification by researchers and are a valuable tool in molecular cloning 11.2 BACKGROUND OF RESTRICTION ENDONUCLEASE The name restriction enzyme has originated from the studies of phage λ and the phenomenon of host-controlled restriction and modification of a bacte- rial virus (Winnacker, 1987).
The process was first recognized in the work done in the laboratories of Salvador Luria and Giuseppe Bertani in early 1950s (Luria and Human, 1952; Bertani and Weigle, 1953). It was found that a bacteriophage λ which can grow well in one strain of bacteria, such as Escherichia coli K, when allowed to grown in another strain, such as E. coli C, its yields can drop significantly. The host cell, E.
coli C, is called as the restricting host and have the capability to decrease the phage activity. If a phage λ grown in one strain, the ability of that phage to grow in the other strains also becomes restricted. In the 1960s, Werner Arber and Matthew Meselson showed that the restriction was instigated by an enzymatic break- down of the phage λ DNA. The enzyme involved in the breakdown of phage DNA was coined as a restriction enzyme (Meselson and Yuan, 1968; Dussoix and Arber, 1962; Lederberg and Meselson, 1964).
The restriction endonuclease studied by Arber and Meselson were type I restriction enzymes, which cleaves DNA randomly away from the recog- nition site. The isolation and characterization of the first type II restriction Restriction Endonucleases 239 enzyme, HindII, from the bacterium Haemophilus influenzae was carried out by Hamilton O. Smith, Thomas Kelly, and Kent Wilcox in 1970. The type II restriction enzymes are more useful for laboratory use, as they cut the DNA within their recognition sequence.
Later, Daniel Nathans and Kath- leen Danna showed that the cleavage of simian virus 40 (SV40) DNA by restriction enzymes produce particular fragments which can be separated by polyacrylamide gel electrophoresis. This result showed that the restriction enzymes can also be useful in the mapping of the DNA (Danna and Nathans, 1971). For this work, Werner Arber, Daniel Nathans, and Hamilton O. Smith was awarded the 1978 Nobel Prize in Physiology or Medicine.
The innova- tion of restriction enzymes paved the way of DNA manipulation, resulting in the development of recombinant DNA technology, which has various appli- cations such as the large scale production of proteins, such as human insulin used by diabetics. The discovery of restriction endonucleases was an impor- tant discovery for predicting the DNA structure and function that further became a backbone for molecular biology studies (Szybalski et al.3 RECOGNITION SITES OF RESTRICTION ENDONUCLEASE Restriction enzymes identify a specific sequence of nucleotides and make a double-stranded cut in the DNA. The recognised DNA sequences can be classified by the total number of bases in its recognition site, usually between 4 and 8 bases. Also, the number of bases in the sequence that deter- mines how often the site will appear in any given genome.
For example, a 4-bp sequence would theoretically occur once every (4)4 or 256 bp, 6 bases at every (4)6 or 4096 bp, and 8 bases at every (4)8 or 65,536 bp. Most of the sequences recognized by restriction enzymes are palindromic sequences. The base sequence that reads the same forward and backward is called as a palindromic sequence. Theoretically, there are two types of palindromic sequences possible in DNA.
First, the mirror-like palindrome that is similar to those found in the ordinary text, in which a sequence reads in the same manner forward and backward on a single strand of DNA strand, e. The second is inverted repeat palindrome that reads the sequence same forward and backward; however, the forward and backward sequences are present in complementary DNA strands (i., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG). The inverted palindromes are more common than mirror-like palindromes. EcoRI digestion produces “sticky ends,” GAATTC, whereas SmaI restric- tion enzyme cleavage produces “blunt ends,” CCC/GGG.
The recognition 240 Plant Biotechnology: Volume 1 sequences in DNA differ for each restriction enzyme, producing DNA of different length and sequence, as well as they differ in their strand orienta- tion (5′ end or the 3′ end). The cut end can be a sticky end “overhang” or blunt end for an enzyme restriction. The restriction enzymes that recognize the same DNA sequence are known as neoschizomers. These often cleave in different locations of the sequence.
However, different enzymes that have recognition and cleavage sequence in the same location are known as isoschizomers. It is known that chromosomes are huge biomolecules that have many genes, and to locate a specific gene physically or manipulate them was impossible before the invention of restriction endonucleases. Previously, scientist isolated and purified the bacterial chromosomes that contain many genes. They used to break the chromosome into smaller segments using physical force that resulted in a random break in the chromosomes and cloned these fragments randomly.
So, for many years, physical manipula- tion of DNA was virtually impossible. It was initially known due to their ability to breakdown/restrict foreign DNA. Restriction enzymes appear to be made exclusively by prokaryotes. It can detect the foreign DNA very easily, such as infecting bacteriophage DNA, and protect the cell from invasion by cleavage of foreign DNA into small pieces making them nonfunctional.
There are multiple functions performed by the restriction enzymes, which cut the DNA/RNA of foreign viruses invading bacteria DNA or DNA/RNA of any of the types of organism. This make them as important and useful tools for molecular genetics. It is generally accepted that restriction enzymes are remarkable tools for the biologists for their investigations in gene orga- nizations, function, and expression. Beside the wide applications of restric- tion enzymes, the structures and catalytic dynamics and mechanism are a hot topic of research for future development (Bourniquel and Bickle, 2002; Mark et al., 1996; Roberts et al., 2003; Titheradge et al.4 DISCOVERY OF RESTRICTION ENZYMES OR RESTRICTION ENDONUCLEASES Restriction enzymes were discovered in 1970, and Werner Arber, Hamilton Smith, and Daniel Nathans received the 1978 Nobel Prize for the discovery (Dussoix and Arber, 1962; Linn and Arber, 1968; Loenen et al.
Restriction enzymes cleave DNA at a specific recognition site and have many uses in molecular biology, genetics, and biotechnology. More than 4000 restriction enzymes are known today, of which more than 621 are Restriction Endonucleases 241 commercially available (Avery et al. The first restriction enzyme isolated was Hind II, but many other restriction enzymes were discovered and characterized later (Kelly and Smith, 1970; Smith and Wilcox, 1970). Restriction enzyme for the first time originated from the studies of phage λ.
The discovery of the restriction endonucleases permits researchers to cleave DNA at specific sites, which is a great benefit over chemical or physical cleavage that results in random fragmentation of DNA. Berg developed a revolutionary idea to create recombinant DNA for the first time in 1972. Restriction endonucleases are mostly present in bacteria. However, their presence has been confirmed in archaebacteria, viruses, and even in eukary- otes.
The discovery of restriction enzymes paved the way for scientists to cut the DNA into specific pieces. Every time a given piece of DNA was cut with a given enzyme, the same fragments were produced. These defined pieces could be put back together in new ways. So, in conclusion, cutting DNA molecules in a particular region and reproducible order opened new gate of experimental possibilities.5 TYPES OF RESTRICTION ENDONUCLEASES The naturally occurring restriction endonucleases are divided into three main groups (types I, II, and III), depending on their enzyme cofactor require- ments, composition, nature of their target sequence, and the position of their DNA cut-site relative to the target sequence.
However, type IV and type V are also reported (Bickle and Krüger, 1993; Boyer, 1971; Yuan, 1981).