Activation of DNA Cleavage by Oligomerization of DNA-Bound SgrAI

Probing the Run-On Oligomer of Activated SgrAI Bound to DNA

SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single bas...

Cleavage of DNA by Mammalian DNA Topoisomerase

Using the P4 unknotting assay, DNA topoisomerase I1 has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase I1 can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerse 11-DNA complex and is reversible with respect to salt and temperature...

The structure of SgrAI bound to DNA; recognition of an 8 base pair target

The three-dimensional X-ray crystal structure of the 'rare cutting' type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca(2+) bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn(2+), with strong ...

Domain Swapping in Allosteric Modulation of DNA Specificity

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA ...

, “ Dna Cleavage by Hydrolysis ”