Engineering a nicking endonuclease N.AlwI by domain swapping

Changing enzymatic function through genetic engineering still presents a ch allenge to molecular biologists. Here we present an example in which changi ng the oligomerization state of an enzyme changes its function. Type Ils re striction endonucleases such as AlwI usually fold into two separate domains : a DNA-binding domain and a catalytic/dimerization domain. We have swapped the putative dimerization domain of AlwI with a nonfunctional dimerization domain from a nicking enzyme, N.BstNBI. The resulting chimeric enzyme, N.A lwI, no longer forms a dimer. Interestingly, the monomeric N.AlwI still rec ognizes the same sequence as AlwI but only cleaves the DNA strand containin g the sequence 5'-GGATC-3' (top strand). In contrast, the wild-type AlwI ex ists as a dimer in solution and cleaves two DNA strands; the top strand is cleaved by an enzyme binding to that sequence, and its complementary bottom strand is cleaved by the second enzyme dimerized with the first enzyme. N. AlwI is unable to form a dimer and therefore nicks DNA as a monomer. In add ition, the engineered nicking enzyme is at least as active as the wild-type AlwI and is thus a useful enzyme. To our knowledge, this is the first repo rt of creating a nicking enzyme by domain swapping.