Interaction of restriction and modification enzymes on their recognition sequences

Abstract

DNA-protein interactions have examined by comparing the action of the two enzymes, endonuclease and methylase, on their recognition sequence, and sequences containing modified bases. Two restriction-modification system, BamHI and Clal, have been examined for this study. Thymine was replaced by uracil and 5-bromouracil to determine the role of 5-methyl group located in major groove in recognizing the specific sequence. Guanine was replaced by hypoxanthine to determine the role of 2-amino group located in minor groove in recognizing the specific sequence. The 5-methyl group of the T residue appeared to interact strongly with the BamHi endonuclease since the enzyme could not cleave the dodecanucleotide (GACGGAUCCGTC). However the BamHi methylase could recognize the sequence containing dU residue although the reaction rate was significantly slowed down. 5-bromine could not compensate the role of 5-methyl group at all. When the 2-amino group of the first guanidine residue was removed (IGATCC) the BamHi endonuclease activity was slowed down while the BamHi methylase activity was completely disappeared. The 2-amino group of the second guanidine residue turned out to be essential for the BamHi endonuclease activity while the sequence GIATCC was normally recognized by the BamHi methylase. These results indicate that the ways recognizing their sequence by BamHi endonuclease and BamHi methylase are different. The 5-methyl group of the first T residue at position 2 appeared to interact strongly with the Clal methylase since the enzyme could not methylate the dodecanucleotide (GACAUCGATGTC). However the Clal endonuclease could recognize the sequence containing dU residue although the reaction rate was significantly slow down. The 5-methyl group of the second T residue at position 6 also appeared to interact strongly with Clal methylase. In spite of very slow rate, Clal endonuciease could cleave the dodecanucleotide containing this modified base. 5-bromine of the secon...