MACROEVOLUTION BY TRANSPOSITION - DRASTIC MODIFICATION OF DNA RECOGNITION BY A TYPE-I RESTRICTION ENZYME FOLLOWING TN5 TRANSPOSITION

We have characterized a novel mutant of EcoDXXI, a type IC DNA restric tion and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enz ymes, the wild type EcoDXXI recognizes a sequence composed of two asym metrical half sites separated by a spacer region: TCA(N7)RTTC. Purific ation of the EcoDXXI mutant methylase and subsequent in vitro DNA meth ylation assays identified the mutant recognition sequence as an interr upted palindrome, TCA(N8)TGA, in which the 5' half site of the wild ty pe site is repeated in inverse orientation. The additional base pair i n the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Seque ncing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain w hich recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain an d the conserved repeated sequence that defines the length of the recog nition site spacer region. We propose that the EcoDXXI mutant methylas e utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.