Engineering a nicking endonuclease N. Alw I by domain swapping

Changing enzymatic function through genetic engineering still presents a challenge to molecular biologists. Here we present an example in which changing the oligomerization state of an enzyme changes its function. Type IIs restriction endonucleases such as Alw I usually fold into two separate domains: a DNA-binding domain and a catalytic/dimerization domain. We have swapped the putative dimerization domain of Alw I with a nonfunctional dimerization domain from a nicking enzyme, N. Bst NBI. The resulting chimeric enzyme, N. Alw I, no longer forms a dimer. Interestingly, the monomeric N. Alw I still recognizes the same sequence as Alw I but only cleaves the DNA strand containing the sequence 5′-GGATC-3′ (top strand). In contrast, the wild-type Alw I exists 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. Alw I is unable to form a dimer and therefore nicks DNA as a monomer. In addition, the engineered nicking enzyme is at least as active as the wild-type Alw I and is thus a useful enzyme. To our knowledge, this is the first report of creating a nicking enzyme by domain swapping.