Model for how type I restriction enzymes select cleavage sites in DNA.

Abstract

Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. AU details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (is) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37TC indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can be further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity ofEcoB and EcoK, and it explains in a simple way how cleavage sites are selected.The type I restriction enzymes EcoB and EcoK have a complex mode ofaction (reviewed in refs. 1-3). They act only on double-stranded DNA that contains a unique recognition sequence, TGAN8TGCT for EcoB and AACN6GTGC for EcoK (N = any nucleotide). Specific binding to these sites requires S-adenosylmethionine, and further reactions require (or, in the case of methylation, are stimulated by) ATP. In the presence of ATP, the state of methylation of the recognition sequence determines the course of the reaction: if both strands are methylated, the enzyme falls off the DNA; if one strand is methylated, the enzyme rapidly methylates the second strand; if neither strand is methylated, the enzyme hydrolyzes large amounts of ATP, translocates considerable lengths ofDNA, and cuts the DNA at seemingly random sites far from the recognition sequence. In the nucleolytic mode, the enzyme is used up in the reaction, apparently remaining bound at its recognition site. The effect of this complex set of reactions is to maintain resident DNA intact but to degrade unmethylated foreign DNA.One puzzling aspect of the nuclease activity of EcoB and EcoK has been how cleavage sites are selected in the DNA. We believe we have now discovered how this is done.

Immigration control of DNA in bacteria: self versus non-self

“…Experiments in vivo, using phage DNA substrates for restriction by EcoKI (Brammar et al, 1974 ;Garcia & Molineux, 1999), support the model (Studier & Bandyopadhyay, 1988) in which cutting occurs between two target sequences when the translocating complexes collide. It is not known whether any feature of the structure or organization of the nucleoid, or any process such as DNA replication, would either reduce the speed of DNA translocation, or alternatively halt translocation and stimulate endonuclease activity.…”

“…Our current understanding of the mode of action of a type I restriction enzyme is essentially as outlined in the ' collision ' model of Studier & Bandyopadhyay (1988). According to this model (Fig.…”

“…target sequence, translocates the DNA from both sides towards itself in an ATP-dependent manner. DNA cleavage occurs when translocation is impeded (Studier & Bandyopadhyay, 1988 ;Janscak et al, 1999a). The three subunits of a type I R-M system are encoded by closely linked genes : hsdR, hsdM and hsdS.…”

“…The model for DNA breakage described by Studier & Bandyopadhyay (1988). EcoKI bound to target sequences translocates DNA towards itself.…”

Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications

“…Three types of DNA restriction systems are presently known: I, II, and III. Because type I restriction enzymes, more precisely restrictionmodification (R-M) systems, recognize a specific sequence but cleave randomly far from the recognition sequence, they are distinguished from type II and III enzymes that recognize and cleave specific target DNA sequences (1,2). The type I enzymes are heterogeneous complexes, consisting of a specificity subunit (S-subunit) that is responsible for recognizing a specific DNA sequence, a methylation subunit (M-subunit) that methylates the target adenine nucleotides recognized by the S-subunit, and a restriction subunit (R-subunit) that randomly cleaves DNA (3,4).…”