Restriction Enzymes

Enzymes that cut the phosphodiester bonds of a polynucleotide chain are called nucleases, and they come in two types: exonucleases, which remove nucleotides from the ends of a DNA strand, and endonucleases, which cut DNA from within the strand. During the 1970s, scientists discovered that bacteria contain nucleases that recognise a short, specific nucleotide sequence within duplex DNA and cut it there -- these are called restriction endonucleases (REN) or simply restriction enzymes (RE). They get this name because bacteria use them to destroy the DNA of invading viruses, thereby 'restricting' the virus's ability to grow inside the cell; the bacterium protects its own DNA from the same fate by methylating the bases at the susceptible sites, which chemically blocks the enzyme's action there. Restriction enzymes are, in effect, molecular scissors that recognise and cut DNA at specific sequences, called recognition sequences or recognition sites; Table 12.2 lists a few examples (AluI, BamHI, EcoRI and HindII) along with their bacterial source and the sequence each one cuts.

Recognition sequences are typically 4 to 8 nucleotides long, and are characterised by a particular kind of internal symmetry called a palindrome -- a sequence that reads identically whether you read the top strand 5' to 3' or the bottom strand 5' to 3'. EcoRI, for example, recognises the palindromic sequence GAATTC. When EcoRI (or a similar enzyme) cuts a palindrome at an offset point on each strand rather than straight across, it produces short single-stranded overhangs at each end of the DNA -- called cohesive, sticky, or staggered ends (Fig 12.3 shows this for EcoRI). If the enzyme instead cuts straight across the palindrome, it produces flush blunt ends instead (as AluI and HindII do, per Table 12.2). …