Type III restriction enzymes communicate in 1D without looping between their target sites.

To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA.

Subunit assembly modulates the activities of the Type III restriction-modification enzyme PstII in vitro.

We demonstrate that, like other Type III restriction endonuclease, PstII does not turnover such that a DNA substrate is only fully cleaved at a Res2Mod2-to-site ratio of approximately 1:1. However, unlike other Type III enzymes, the cleavage rate profiles varied with protein concentration: using 5 nM DNA and 25 nM PstII, approximately half of the DNA was cut at a fast rate while the remainder was cut 24 times more slowly; in comparison, with 100 nM PstII cleavage occurs at a single fast rate. The inclusion of the methyl donor S-adenosyl methionine does not alter the rates with 100 nM PstII but with 25 nM PstII the reaction stopped after completion of the initial fast cleavage phase owing to methylation. Concentration-dependent rates were also observed in methylation assays: at 100 nM PstII, a single slow rate was measured while at lower PstII concentrations both fast and slow rates were measured. We propose a model in which the intact Res2Mod2 complex favoured at high PstII concentrations is a fast endonuclease/slow methyltransferase while the various subassemblies which coexist at lower concentrations are fast methyltransferases. A potential role for disassembly in control of restriction activity in vivo is discussed.

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii.

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5'-CTGATG(N)(25-26/27-28)-3'. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis ( approximately 40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25-26 nt downstream of the top strand sequence to generate a two base 5'-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5'-CATCAG-3'. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein-protein contacts to activate endonuclease activity.