Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme

Abstract

Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25-27 bp downstream of one of the two sites. The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation. The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme. In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site. Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site. Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates. Such cleavage can be abolished by the binding of Lac repressor downstream (3' side) but not upstream (5' side) of the recognition site. Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity. In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5' to 3' direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends. This study demonstrates a new facet in the mode of action of these restriction enzymes.

Figure 1

Identical and non-identical sequence context of recognition sites and R.EcoP15I activity. (A) Schematic representation of pJA31 substrate. Head-to-head oriented recognition sites A and A′in identical sequence context. (B) Representation of pJA24. Recognition sites A and D in non-identical sequence context. In both (A) and (B), the distances between consecutive sites are given in kb or bp. Recognition sites are symbolized by a black triangle representing 5′-CAGCAG-3′. Base of the triangle is 5′ and tip of triangle is 3′ end of recognition site. Lac rep binding site is represented by a hatched square. The 1.2 kb fragment described in the text is marked by a bracket. (C) R.EcoP15I cleavage of circular pJA31 and pJA24. Lanes 1 and 4, digestions of pJA31 and pJA24 with R.EcoP15I. Lanes 2 and 3, undigested pJA31 and pJA24 substrates, respectively. Lane M, DNA marker bands, sizes are given in kb between gel pictures. Sizes of R.EcoP15I digested bands are given in kb next to respective gel pictures.

Figure 2

Cleavage of sites proximal to DNA end. (A) Substrates having non head-to-head oriented sites. Symbols as in Figure 1. Sites A and D have the 3′ side of the recognition site towards the free DNA end. (B) R.EcoP15I cleavage pattern of AvaII-digested pJA24 DNA. Lane 1, AvaII-digested pJA24 DNA; lanes 2–5, AvaII-digested pJA24 DNA incubated with increasing amounts of R.EcoP15I, respectively. (C) R.EcoP15I cleavage of HindIII- or KpnI-digested pJA24 DNA. Lanes 1 and 4, pJA24 DNA digested with HindIII and KpnI, respectively; lanes 2 and 3, HindIII- or KpnI-digested pJA24 DNA incubated with R.EcoP15I enzyme. The lower portion (<1.6 kb) of this gel picture has been intensified and realigned with rest of the picture to increase clarity. In (B) and (C), lane M, DNA molecular weight markers.

Figure 3

R.EcoP15I activity on DNA containing a single recognition site in the absence or in the presence of Lac rep. (A) Single-site DNA substrates generated by digesting pJA24 with AccI (top panel) and HincII (bottom panel). (B) Blocking access to the DNA end on the 5′ side of site A. Lane 1, AccI-digested pJA24 DNA; lane 2, AccI-digested pJA24 DNA incubated with R.EcoP15I; lane 3, same as in lane 2 in the presence of Lac rep; lane 4, same as in lane 3 in the presence of IPTG (0.1 mM). The lower portion (<1.5 kb) of this gel picture has been intensified and shown below. (C) Blocking access to the DNA end on the 3′ side of site A′. Lane 1, HincII-digested pJA24 DNA; lane 2, HincII-digested pJA24 DNA incubated with R.EcoP15I; lane 3, same as in lane 2 in the presence of Lac rep; lane 4, same as in lane 3 in the presence of IPTG (0.1 mM). The lower portion (<1.25 kb) of this gel picture has been intensified and shown below. In (B) and (C), lane M, DNA molecular weight markers.

Figure 4

R.EcoP15I activity in the presence of HU protein. (A) Binding of HU protein to pJA31 DNA. DNA mobility decreases with increasing HU concentration. (B) R.EcoP15I activity in the presence of increasing HU concentration. Lane C, assay in the absence of HU protein; lanes 1, 2, 3 and 4 correspond to reactions having 1, 2, 3 and 4 μg of HU protein, respectively. Lane M, DNA molecular weight markers.

Figure 5

Mechanism of action of Type III restriction enzymes. Interaction of the enzyme with a DNA end. R.EcoP15I recognition site symbolized by ‘▸’. The Mod subunit is represented by ‘1’ and the Res subunit by ‘2’. R₂M₂ is shown as ‘3’, ‘4’ indicates cleavage complex. The arrows indicate the direction of R.EcoP15I/cleavage complex translocation. The box shows events on single-site DNA leading to cleavage complex formation.