Restriction enzymes and their use in molecular biology: An overview.

Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the scientists whose pioneering work had explored the activity and sequence specificity of these enzymes, contributing to the definition of their enormous potential as tools for DNA characterization, mapping and manipulation, were awarded the Nobel Prize. In this short review, we celebrate the history of these enzymes in the light of their many different uses, as these proteins have accompanied the history of DNA for over 50 years representing active witnesses of major steps in the field.

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

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.

Tracking EcoKI and DNA fifty years on: a golden story full of surprises.

1953 was a historical year for biology, as it marked the birth of the DNA helix, but also a report by Bertani and Weigle on 'a barrier to infection' of bacteriophage lambda in its natural host, Escherichia coli K-12, that could be lifted by 'host-controlled variation' of the virus. This paper lay dormant till Nobel laureate Arber and PhD student Dussoix showed that the lambda DNA was rejected and degraded upon infection of different bacterial hosts, unless it carried host-specific modification of that DNA, thus laying the foundations for the phenomenon of restriction and modification (R-M). The restriction enzyme of E.coli K-12, EcoKI, was purified in 1968 and required S-adenosylmethionine (AdoMet) and ATP as cofactors. By the end of the decade there was substantial evidence for a chromosomal locus hsdK with three genes encoding restriction (R), modification (M) and specificity (S) subunits that assembled into a large complex of >400 kDa. The 1970s brought the message that EcoKI cut away from its DNA recognition target, to which site the enzyme remained bound while translocating the DNA past itself, with concomitant ATP hydrolysis and subsequent double-strand nicks. This translocation event created clearly visible DNA loops in the electron microscope. EcoKI became the archetypal Type I R-M enzyme with curious DNA translocating properties reminiscent of helicases, recognizing the bipartite asymmetric site AAC(N6)GTGC. Cloning of the hsdK locus in 1976 facilitated molecular understanding of this sophisticated R-M complex and in an elegant 'pas de deux' Murray and Dryden constructed the present model based on a large body of experimental data plus bioinformatics. This review celebrates the golden anniversary of EcoKI and ends with the exciting progress on the vital issue of restriction alleviation after DNA damage, also first reported in 1953, which involves intricate control of R subunit activity by the bacterial proteasome ClpXP, important results that will keep scientists on the EcoKI track for another 50 years to come.