One recognition sequence, seven restriction enzymes, five reaction mechanisms

Abstract

The diversity of reaction mechanisms employed by Type II restriction enzymes was investigated by analysing the reactions of seven endonucleases at the same DNA sequence. NarI, KasI, Mly113I, SfoI, EgeI, EheI and BbeI cleave DNA at several different positions in the sequence 5'-GGCGCC-3'. Their reactions on plasmids with one or two copies of this sequence revealed five distinct mechanisms. These differ in terms of the number of sites the enzyme binds, and the number of phosphodiester bonds cleaved per turnover. NarI binds two sites, but cleaves only one bond per DNA-binding event. KasI also cuts only one bond per turnover but acts at individual sites, preferring intact to nicked sites. Mly113I cuts both strands of its recognition sites, but shows full activity only when bound to two sites, which are then cleaved concertedly. SfoI, EgeI and EheI cut both strands at individual sites, in the manner historically considered as normal for Type II enzymes. Finally, BbeI displays an absolute requirement for two sites in close physical proximity, which are cleaved concertedly. The range of reaction mechanisms for restriction enzymes is thus larger than commonly imagined, as is the number of enzymes needing two recognition sites.

Figure 1

Enzymes and substrates. (A) Shown, within the grey box, are the sequence GGCGCC and the sequences flanking this site in pUC19. The enzymes KasI, NarI, Mly113I, SfoI, EgeI, EheI and BbeI cleave GGCGCC at the positions shown. (B) The plasmids pUC19 (2686 bp) and pDG5 (3806 bp) contain, respectively, one and two copies of the sequence in (A), indicated by the grey box(es). In pDG5, the two GGCGCC sites are separated by 1063 (or 2743 bp) and are interspersed with two res sites from Tn21 (arrowheads). The action of Tn21 resolvase at the res sites generates a catenane consisting of two interlinked circles of covalently closed DNA, one large (3120 bp) and one small (686 bp). Both circles carry one copy of the GGCGCC sequence (grey box).

Figure 2

NarI on one- and two-site plasmids. Reactions contained 40 U/ml NarI and 5 nM supercoiled (SC) DNA in buffer A at 37°C. The DNA was in (A), pUC19, with one NarI site; in (B), pDG5, with two NarI sites. In both cases, the upper panel shows an agarose gel of samples taken from the reaction at the times indicated: the electrophoretic mobilities of the open-circle (OC), full-length linear (FLL) and SC DNA are marked on the right: in (B), the two linear products from cutting both sites (L1 and L2) are also marked. The lower panels show the concentrations of the following forms of the DNA during the first 60 min of the reaction: SC substrate, black squares; OC DNA, white circles; FLL DNA, black triangles; (in B alone), the mean of the two final products (L1,L2), white inverted triangles.

Figure 3

NarI on catenated DNA. (A) The scheme shows all of the distinct products from cleaving a catenane in one site in each ring: S, O and L denote supercoiled, open-circle and linear DNA respectively. Shown in red; the intact catenane containing two interlinked rings of SC DNA (S_LS_S), one large (3120 bp) and one small (686 bp), marked, respectively, by the subscripts ‘L’ and ‘S’; in blue, the products after cutting one phosphodiester bond, two interlinked rings nicked in either the large (O_LS_S) or the small (S_LO_S) ring; in green, the products after cutting two bonds, either one linear and one supercoiled species (L_L + S_S from cutting both strands in the large ring, or S_L + L_S from cutting both in the small) or two interlinked rings of OC DNA (O_LO_S); in grey, the products after cutting three bonds, one linear and one OC form (L_L + O_S or O_L + L_S); in purple, the large (L_L) and the small (L_S) linear products after cutting all four bonds. (B) The reaction contained 40 U/ml NarI and 5 nM Cat (the catenane from pDG5; Figure 1B) in buffer A at 37°C. Samples from the reactions were analysed by electrophoresis through agarose to separate, wherever possible, all of the species in (A). The concentrations of the following forms were assessed: intact catenane, red squares; the sums of the products cleaved in one (blue circles), two (green triangles), three (grey diamonds) and four (purple inverted triangles) phosphodiester bonds. (In cases where the product contains unlinked species, e.g. L_L + S_S, the mean of the two concentrations is noted.)

Figure 4

KasI on one- and two-site DNA. Reactions contained 40 U/ml KasI and 5 nM SC DNA in buffer B at 37°C. The DNA was in (A), pUC19, with one KasI site; in (B), pDG5, with two KasI sites; in (C), Cat (Figure 1B), with one KasI site in each ring. Samples taken from the reactions were analysed by electrophoresis through agarose. For the reactions in (A) and (B), the following were assessed: SC substrate, black squares; OC DNA, white circles; FLL DNA, black triangles; (in B alone), the mean of the two final products (L1,L2), white inverted triangles. For the reaction in (C), the concentrations of the various products from a catenane with one recognition site in each ring (Figure 2A) were assessed as in Figure 2B. Shown here are the products cleaved at two phosphodiester bonds: the DNA cleaved in one strand at each site (i.e., the catenane containing two interlinked rings of nicked DNA, O_LO_S), black triangles; the sum total of the two species cut in both strands at one site (in either the small ring, to release S_L, or the large ring, to release S_S), white triangles.

Figure 5

Mly113I on one- and two-site DNA at varied salt. Reactions at 37°C contained 5 U/ml Mly113II and 5 nM SC DNA in either buffer A (A and B) or buffer A + 150 mM NaCl (C and D). The DNA was either pUC19, with one Mly113I site (A and C), or pDG5, with two Mly113I sites (B and D). Samples were taken from the reactions at various times and were analysed by electrophoresis through agarose. The concentrations of the following forms of the DNA were assessed: SC substrate, red squares; OC DNA, blue circles; FLL DNA, green triangles; (in B and D) the mean of the two final products (L1,L2), purple inverted triangles.

Figure 6

SfoI on one- and two-site DNA. Reactions contained 12 U/ml SfoI and 5 nM SC DNA in buffer B at 21°C. The DNA was: in (A), pUC19, with one SfoI site; in (B), pDG5, with two SfoI sites. Samples were taken from the reactions at various times and analysed to assess the following: SC substrate, black squares; OC DNA, white circles; FLL DNA, black triangles; [in (B) alone], the mean of the two final products (L1, L2), white inverted triangles.

Figure 7

BbeI on one- and two-site DNA. Reactions in buffer A at 37°C contained 30 U/ml BbeI and 5 nM SC DNA. Samples were taken from the reactions at various times and analysed to assess the residual concentration of the substrate. The substrates were: pUC19, with one BbeI site, black squares; pDG5, with two BbeI sites, white circles; Cat (Figure 1B), with one BbeI site in each ring, black triangles.