Abstract
Type IIs endonucleases recognize asymmetric DNA sequences and cleave both strands at fixed positions downstream of the sequence. Many type IIs enzymes, including BspMI, cleave substrates with two sites more rapidly than those with one site. They usually act sequentially on DNA with two sites, but BspMI converted such a substrate directly to the final products cut at both sites. The BspMI endonuclease was found to be a tetramer, in contrast to the monomeric structures for many type IIs enzymes. No change in subunit association occurred during the BspMI reaction. Plasmids with two BspMI sites were cleaved in cis, in reactions spanning sites in the same DNA, even when the sites were separated by just 38 bp. Plasmids with one BspMI site were cleaved in trans, with the enzyme bridging sites in separate DNA molecules: these slow reactions could be accelerated by adding a second DNA with the recognition sequence. Thus, whereas many type IIs enzymes dimerize before cleaving DNA, a process facilitated by two recognition sites in cis, the BspMI tetramer binds two copies of its recognition sequence before cleaving the DNA in both strands at both sites.
Highlights
The endonucleases from type II restriction-modification systems recognize short DNA sequences, 4 – 8-bp long, and cleave both strands of the DNA at fixed locations [1]
Plasmids with one BspMI site were cleaved in trans, with the enzyme bridging sites in separate DNA molecules: these slow reactions could be accelerated by adding a second DNA with the recognition sequence
Whereas many type IIs enzymes dimerize before cleaving DNA, a process facilitated by two recognition sites in cis, the BspMI tetramer binds two copies of its recognition sequence before cleaving the DNA in both strands at both sites
Summary
Proteins—A strain of Escherichia coli that overproduces the BspMI endonuclease was a gift from I. The supernatant contained the majority of the BspMI that had been added to the slurry, whereas most of the impurities were removed, presumably by adsorption onto the hydroxylapatite. This left preparations in which Ն95% of the protein was BspMI endonuclease, as judged by SDS-PAGE. An aliquot (typically 20 l) of the BspMI endonuclease, diluted to the requisite concentration in the appropriate dilution buffer [10], was added to the 3H-labeled plasmid (usually 5 or 10 nM) in 20 mM HEPES (pH 8.0), 1 mM DTT, 10 mM MgCl2 and the concentration of NaCl as specified for each experiment (usually 40 or 80 mM). At various times after adding the enzyme, 15-l samples were removed from the reaction, mixed immediately with 10 l of stop-mix, and analyzed as described previously [10]
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