Highlights of the DNA cutters: a short history of the restriction enzymes

Abstract

In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

Figure 1.

Schematic representation of the functional roles filled in different ways in R-M systems. The functions served include the following: S, DNA sequence specificity; MT, methyltransferase catalytic activity; M, SAM binding; E, endonuclease; T, translocation. Boxes outline functions that are filled by distinct protein domains. Different colours indicate different functions, while different boxes represent distinct domains. Domains in a single polypeptide are abutted, and those in separate polypeptides spaced apart. The order of domains in a polypeptide may vary—e.g. not all Type IIS enzymes have the cleavage domain at the C-terminus. In some cases, functions are integrated with each other, e.g. S and E functions of Type IIP (striped box); in other cases, separate domains carry them out, e.g. Type IIS. See Table 2 for the complexity and diversity of Type II subtypes. The large families of Type I and Type II systems are currently subdivided in 5 and 11 different groups, respectively. The Type I families are distinguished by homology; the Type II groups are distinguished by catalytic properties rather than sequence homology. Type IV enzymes were initially identified as hydroxymethylation-dependent restriction enzymes and currently comprise a highly diverse family. Two examples are shown with and without a translocation domain.

Figure 2.

Photograph of the participants at the EMBO Workshop on restriction in Leuenberg (Basel), Switzerland, 26–30 September 1972, organized by Werner Arber, who took the picture (Archive Noreen Murray). Supplement S1 contains a list with names of the attendees and the program of this meeting, and puts names to faces as far as the attendees could be identified (from the archives of Noreen Murray and Werner Arber).

Figure 3.

Type II restriction enzymes grouped by cleavage properties. ‘Orthodox’IIP enzymes (e.g. EcoRI, EcoRV) cut at the recognition site. Type IIS cut away from the site (e.g. FokI, BfiI). Type IIB require two recognition sites and cut on the outside (e.g. BplI). Type IIE require two recognition sites, and one of the two sites acts as allosteric effector (e.g. EcoRII). Type IIF require two sites and cut at both sites as a tetramer after bringing the two regions together by looping the DNA (e.g. SfiI). Enzymes such as BcnI act as a monomer, in contrast to most Type II REases that act as dimers. See Table 2 and text for further details.

Figure 4.

Intricate control of restriction in the operons of the Type II R-M systems of PvuII and Esp1396I by controlling C proteins. A small C gene upstream of, and partially overlapping with, R is coexpressed from p_res1, located within the M gene, at low level with R after entry of the self-transmissible PvuII plasmid into a new host, while M is expressed at normal levels from its own two promoters p_mod1 and p_mod2 located within the C gene. A similar C protein operates in Esp1396I, but in this case the genes are convergently transcribed with transcription terminator structures in between, and M is expressed from a promoter under negative control of operator O_R, when engaged by C protein in a manner similar to that of the PvuII system. Briefly, the C protein binds to two palindromic sequences (C boxes) defining operator sites O_R and O_L upstream of the C and R genes. After initial low-level expression of C.PvuII protein from the weak promoter p_res1, positive feedback by high-affinity binding of a C protein dimer to the distal O_L site later stimulates expression from the second promoter p_res, resulting in a leaderless transcript and more C and R protein. The proximal site O_R is a much weaker binding site, but C protein bound at O_L enhances the affinity of O_R for C protein, and at high levels of C protein, the protein-O_R complex downregulates expression of C and R. In this way, C protein is both an activator and negative regulator of its own transcription. In addition, it is a negative regulator of M, which makes sense as overmethylation of DNA may also be harmful to the cell (see text for further details). C.Esp1396I controls O_R, O_L and O_M in a similar manner as described above. In this way, C proteins keep both R-M under control, and have been tentatively identified in >300 R-M systems (Table 2).