1. page: Genial - Restriction Enzymes and Site-Specific DNA Cleavage Procedures for chemical isolation of DNA 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. The most commonly used 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. Technically, the enzymes are known as type II restriction endonucleases. The restriction enzyme BamH I is one example of several hundred restriction enzymes that are known. It recognises the double- stranded sequence 5’-GGATCC-3’ 3’-CCTAGG-5’ and cleaves each strand between the G-bearing nucleotides marked in bold. A DNA sequence with this type of symmetry is called a palindrome. In ordinary English, a palindrome is a word or phrase that reads the same forwards and backwards, such as “ABBA” or “OTTO” or “MADAM” (Hartl and Jones 2000, slightly changed). History Restriction enzymes are responsible for the phenomenon in bacteria known as host-controlled restriction modification or phenotypic modification. Bacteriophage (phage) are viruses which infect bacteria and take over the host's nucleic acid synthesis machinery in order to replicate the phage genome. After multiplying to several hundred copies, the host is killed and the progeny phage are released. In the early 1950s, Luria et al. observed that phage which easily infect one strain of bacteria often failed to generate plaques on a second bacterial strain, the phage was "restricted" to one strain of bacteria. In 1962, Arber and Dussoix proposed a molecular model to explain the "restriction" phenomenon. They postulated that certain bacterial strains contained endonucleases able to cleave unprotected DNA, and other strains contained a modification system responsible for protecting their own DNA. Phage DNA was also modified by the host replication machinery. Unprotected DNA was degraded by the endonuclease which restricted infection by unmodified phage, hence the term restriction endonuclease. In 1968, Arber and Linn demonstrated the "restriction" endonuclease activity of the EcoB restriction enzyme and Meselson and Yuan purified a similar enzyme from E. coli K. These enzymes were able to cleave DNA from other strains but not that of the original strains. These restriction enzymes cleaved DNA at random positions, often far removed from the recognition site. In 1970, Smith, Wilcox and Kelly described the purification and characterization of the recognition and cleavage site of a more useful restriction enzyme, Hind II. This enzyme cut the same site it recognized, and was used by Smith's colleague Daniel Nathans to cut the circular genome of simian virus 40 into 11 specific fragments and to generate the first restriction maps (Promega 1996, slightly changed). Most restriction enzymes are named after the species in which they were found. BamH I, 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 italicised. The DNA fragment produced by a pair of adjacent cuts in a DNA molecule is called a restriction fragment. A large DNA molecule will typically be cut into many restriction fragments of different sizes. For example, an E. coli DNA molecule, which contains 4.6 x 10 6 base pairs, is cut into several hundred to several thousand fragments and mammalian genomic DNA is cut into more than a million fragments. Although these numbers are large, they are actually quite small relative to the number of sugar-phosphate bonds in the DNA of an organism. 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 recognised for cleavage by a restriction enzyme is called the restriction site of the enzyme. The restriction sites of most commercially used restriction enzymes are palindromes (Hartl and Jones 2000). Worksheet IX.4 Restriction enzymes