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

Abstract

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.

Figure 1

The Bertani and Weigle experiment (1953). The discovery of a barrier to infection of λ in its natural host, E.coli K-12, if the phage had been previously propagated on E.coli C. This led to the phenomenon of R-M. Escherichia coli K-12 possesses a R-M system, EcoKI, absent in E.coli C. Phage λ propagated on E.coli C (called λ.C or λ.0) is not protected from restriction by EcoKI and forms plaques with low efficiency (e.o.p.) on E.coli K-12 as compared to E.coli C (bold). Pretreatment of cells with UV leads to RA (italic). The mechanism behind this phenomenon was recently elucidated by Svetlana Makovets and Noreen Murray.

Figure 2

Tracking EcoKI, Nobel Laureate Werner Arber and Noreen Murray.

Figure 3

Evolution of new Type I specificities via domain shuffling of the N-terminal and C-terminal TRD (a), or alterations in the length of the spacer between the two TRDs (b). Black regions indicate conserved regions involved in subunit interactions and determine the length of the spacer between the two recognition sequences. (a) StySQ is an in vivo recombinant of StySPI and the enzymes share the 5′AAC-recognition domain (red). StySPI shares the C-terminal domain with StySJ (blue), StyLTIII and StySJ the N-terminal GAG-recognition domain (green) and finally StyLTIII and StySQ both recognize RTAYG 3′ (orange). (b) EcoR124I differs from EcoR124II in a TAEL tetrapeptide in the central conserved region, which is repeated two and three times, respectively, resulting in an extra base in the spacer between the tri- and tetranucleotides of the recognition sequence. EcoDXXIsI results from a transposon insertion in hsdS of EcoDXXI, leading to a truncated polypeptide. Such a polypeptide functions as a dimer on a symmetrical trinucleotide sequence with a specificity dictated by the single TRD.

Figure 4

Model of the structure of the EcoKI restriction complex [adapted from Davies et al. (95), reproduced with permission from Elsevier. Copyright 1999]. The two TRDs of the specificity subunit HsdS (green) recognize the two halves of the DNA recognition site AAC(N6)GTGC. The TRDs are linked by conserved sequence regions which function as subunit interfaces and also define the length of the non-specific DNA sequence in the middle of the recognition site. Two HsdM modification subunits (blue) bind to the conserved regions of HsdS via their N- and C-terminal domains. They wrap around the DNA helix on the opposite side of S, allowing access of the methyltransferase domain of M to DNA, presumably using base flipping as described for Type II MTases. Two HsdR subunits (orange) associate with M and S via the C-terminus. The central part of the protein is involved in translocation and contains ‘DEAD box’ motifs, characteristic of helicases (H). These motifs probably fold into two domains (IA and 2A) to form a cleft through which the DNA would pass (resembling a ‘RecA-like’ structure that may be common to all helicases/translocases). EcoKI belongs to helicase superfamily 2, whose members are believed to guide the DNA via regions outside the IA and IIA domains towards the cleft involving interactions with the DNA backbone (and not the bases), in line with the function of EcoKI as a DNA translocase rather than helicase. In the N-terminus of HsdR is a motif, characteristic of endonucleases (R). The enzyme binds the target site via HsdM and HsdS using AdoMet as cofactor for binding and distinguishing between hemimethylated and unmodified DNA. If unmodified, the enzyme undergoes a large conformational change and translocates the DNA past itself, while remaining bound to the recognition site, creating large loops visible by EM and AFM, concomitant with ATP hydrolysis. The model rests on extensive genetic, biochemical and biophysical evidence (see text for further details and references). (A) A model of amino acids 43–157 from the N-terminal TRD of EcoKI interaction with DNA [reproduced with permission from Sturrock,S.S. and Dryden,D.T.F. (1997) Nucleic Acids Res., 25, 3408–3414; 79]. (B) A front view from a partial model of a Type I MTase bound to DNA constructed using two copies of the structure of Type II MTases bound to DNA [from Dryden et al. (77) with permission from Nature Publishing Group (http://www.nature.com/nsb/)]. The TRD regions are based on the structure of the TRD from HhaI and the methyltransferase domains in the catalytic domains of TaqI. Space filling shows sites of mutations resulting in loss of specificity and activity. (C) Section of the HsdR subunit showing mutational analysis of conserved endonuclease and ‘DEAD box’ (helicase-like) motifs [from Davies et al. (89), reproduced with permission from Elsevier. Copyright 1999].

Figure 5

Model for the mechanism of ClpXP-dependent control of restriction. When EcoKI binds to an unmodified chromosomal target sequence, translocation starts. However, ClpXP recognizes the R subunit in the translocation-proficient state and destroys it, thereby preventing further translocation and cutting of the chromosome [from Murray (3) with permission from the American Society for Microbiology. Copyright 2000].