Restriction Enzymes

A restriction enzyme (restriction endonuclease) is, in essence, a pair of molecular scissors: it recognises a short, specific DNA sequence (the restriction site) and cleaves the DNA there, and nowhere else. The discovery traces back to 1963, when researchers working with bacteriophage infection of E. coli isolated two enzymes acting on the phage's DNA - one that added protective methyl groups, and a separate one that actually cut the DNA, which came to be called the restriction endonuclease. This is not an isolated laboratory curiosity: bacteria naturally carry these enzymes as part of a restriction-modification defence system, methylating and thereby protecting their own DNA while cutting up any unmethylated (i.e. foreign, viral) DNA that enters the cell. Restriction enzymes split first by mode of action into exonucleases, which nibble nucleotides away one at a time starting from a DNA molecule's free end (examples: Bal 31, Exonuclease III), and endonucleases, which instead cut internal phosphodiester bonds somewhere in the middle of the molecule (examples: Hind II, EcoRI, PvuI, BamHI, TaqI) - a distinction that matters because it is exactly the pairing tested in the matching-type question later in this chapter's evaluation. There are also three broader classes - Type I, Type II and Type III - that differ in their precise mechanism, but only Type II enzymes are actually used in recombinant DNA technology, because they cut cleanly within a short, usually palindromic, 4-8 base-pair recognition sequence rather than cutting at some variable distance away from it. Today over 900 distinct restriction enzymes have been catalogued from more than 230 different bacterial strains. Naming follows a fixed convention worth knowing cold, since it is directly tested: the first letter is the genus, the next two letters are the species, an optional letter/number gives the bacterial strain, and a Roman numeral records the order of discovery within that organism - so EcoRI is the first restriction endonuclease discovered in Escherichia coli strain RY13. Finally, how an enzyme cuts a recognition site matters for downstream cloning: some cut straight thr …