DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes

Abstract

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

Figure 1

EcoP15I–DNA complexes in the presence of ATPγS. (A) Complexes on single-site template SS in high-salt buffer. Scale bars, 500 nm in main image and 250 nm in montage images. (B) Histogram showing the position of the EcoP15I relative to the nearest DNA end.

Figure 2

EcoP15I–DNA complexes in the presence of ATP on one site template. (A, B) Complexes on single site template SS in high-salt (A) and low-salt (B) buffer. Scale bars, 500 nm in main image and 250 nm in montage images. (C–F) Histograms showing the contour length of DNA loops formed by EcoP15I in the presence of ATP (C), the number of loops formed by complexes exhibiting looping (D), position of the EcoP15I and loop origin relative to the nearest DNA end (E) and the loop apex angle (F).

Figure 3

EcoP15I–DNA complexes in the presence of ATP on two-site template. (A) Complexes on two-site template HH and derivatives, in low-salt buffer. Scale bars, 500 nm in main image and 250 nm in montage images. (B) Montage of looped complexes on template HH and HT. (C–E) Histograms showing the contour length of DNA loops formed by EcoP15I on template HH and HT (C), the number of loops formed by complexes exhibiting looping on template HH and −+ (D), position of the EcoP15I and loop origin relative to the nearest DNA end on template HH (E).

Figure 4

The Lac repressor blocks DNA cleavage by EcoP15I. (A) Lac–DNA complexes on template HH in Lac buffer. (B) EcoP15I–DNA complexes in Lac buffer in the presence of ATP on template HH. (C) EcoP15I–Lac-DNA complexes in Lac buffer in the presence of ATP on template HH. Scale bar=500 nm. (D) Lac repressor assay on plasmid pTYB1. The five EcoP15I sites are marked on the map as filled arrows and the Lac-binding sites as rectangles. M, molecular weight marker; lane 1, DNA+ATP; lane 2, DNA+ATP+EcoP15I; lane 3, DNA+ATP+EcoP15I+Lac; lane 4, DNA+ATP+EcoP15I+Lac+IPTG; lane 5, DNA+ATP+EcoP15I+Lac (2 × concentration).

Figure 5

Comparison of loop contour length with a polymer physics model. Histogram represents data for templates SS, HT and HH, pUC19 plasmid DNA and the published data of Reich et al (2004). The line shows the probability distribution for the tear-drop model scaled (vertically) to match the histogram of experimental data. (Yamakawa and Stockmayer, 1972; Sankararaman and Marko, 2005).

Figure 6

Structural model for DNA cleavage by EcoP15I. (A) The structure of the Mod₂Res₂ tetramer bound to the recognition sequence. The Res subunits are shown as red spheres, the Mod subunits as green rectangles (the light green portion represents the catalytic methylation domain and the darker green portion represents the TRD). The left most TRD is responsible for reading the top strand and the right most the lower strand. The yellow arrow represents the EcoP15I-recognition sequence and the purple line represents the DNA cleavage site 25 bp from the adenine in the recognition sequence on the top strand and 27 bp on the bottom strand. The DNase I footprints on each strand in the presence and absence of ATP are shown in blue (Mucke et al, 2001). On the right, the proposed dimer of EcoP15I bound to two copies of the recognition sequence is shown. The rear molecule is semitransparent to allow the DNA and target site to be seen. (B) Cleavage of a head-to-head two-site template via DNA looping and translocation accomplished by the EcoP15I tetramer. Each Res subunit has a DNA-binding site allowing two loops per tetramer labelled 1, 2, 4 and 5, and a loop (labelled 3) resulting from the dimerisation of the tetramers (we have not shown the dimerisation for clarity). By this mechanism, the intersite distance is reduced before the enzymes are brought together by translocation. The leading Res subunits from each tetramer (shown enlarged) then cooperate in double-strand DNA cleavage. DNA segments within the loop caused by 3D looping are shown in blue and those caused by translocation are shown in orange. (C) The influence of dimerisation of EcoP15I on the DNA loops formed by head-to-head-cleavage competent substrates and head-to-tail cleavage-defective substrates. The translocating Res subunits are shown enlarged. Translocation leading to contraction of loop 3 in the head-to-head complex pulls the loop taut between the two translocating Res subunits and we postulate that this is the trigger for cleavage. Translocation of loop 3 in the head-to-tail complex pulls loop 3 taut across the whole complex and we postulate that this does not trigger cleavage.

Figure 7

Search strategies used by restriction enzymes requiring two target sites for cleavage. Type I R/M enzymes only use 1D ATPase-driven processive translocation to close the distance between sites and to ensure correct orientation of the DNA in the cleavage complex. One enzyme binds to each of the two sites. Type IIE and IIF enzymes bind to a single site and then rely upon 3D diffusional looping to bring a second segment of DNA into contact. Upon loop formation, a limited amount of 1D diffusional sliding takes place. If a second target sequence is located during this sliding, before break down of the loop, then cleavage occurs. Type III R/M enzymes combine elements of both type I and type IIE/IIF enzymes. Two copies of the enzyme bind to target sequences on the DNA. 3D diffusional looping occurs bringing the two enzymes closer together. ATPase-driven translocation may start before the loops break down and this brings the two enzymes closer together until collision and cleavage occur.