Effects of base analogue substitutions of the sequence, d(CCGG), on the cleavage and methylation reactions of Hpall and Mspl endonucleases and their cognate methylases

Abstract

The four different enzymes, Hpall endonuclease, Hpall methylase, Mspl endonuclease, and Mspl methylase, were used to understand their specific interactions with the common recognition sequence, CCGG. The synthesized 6 derivatives of the oligonucleotide, AGCCCGGGCT, contains a variety of single base analogue substitutions within the tetrameric recognition core. Cytosine was replaced by 5-methylcytosine or 5-bromocytosine to determine whether these substituted groups affect enzyme-substrate interactions in the major groove. Guanine was replaced by inosine to determine the role of the 2-amino group located in the minor groove. Those substitutions resulted in altered reactivity of the enzymes. Steady state kinetic values were determined for normal and modified oligonucleotides. The addition of 5-bromine or 5-methyl group interfered with the interactions between these enzymes and substrates in the major groove except that Mspl endonuclease could cleave the sequence even with the 5-methyl group at the second cytosine position. The data suggest that there are close contacts between C5 positions of both cytosine residues and these enzymes except that Mspl endonuclease has a space for a methyl group at the C5 position of the second cytosine residue. Hpall or Mspl endonuclease was not able to cleave the oligonucleotides containing inosine nucleotide at either guanine position. The data presented in this thesis also showed that minor groove interactions between 2-amino groups of both guanine residues and both enzymes were essential for the reactions. However, these interactions were not essential for both methylases except that Mspl methylase was not able to methylate the oligonucleotide substituted with inosine nucleotide at the first guanine position.