Effects of base analog substitutions in the sequence, CCGG, on the cleavage and methylation reactions of HpaII and MspI endonucleases and their cognate methylases.
HpaII endonuclease, HpaII methylase, MspI endonuclease, and MspI methylase were used to investigate their specific interactions with the common recognition sequence, CCGG. Six derivatives of the oligonucleotide, AGCCCGGGCT, containing a variety of single base analog substitutions within the tetrameric recognition core were synthesized. Steady state kinetic values for the reactions of all 4 enzymes with these oligonucleotide substrates were obtained. Our data suggest that there are close contacts between the C5 positions of both cytosine residues and the enzymes except that MspI endonuclease can accommodate a methyl group at the C5 position of the second cytosine residue. The data also showed that minor groove interactions between the 2-amino group of both G residues and the HpaII or MspI endonuclease were essential for activity. However, these interactions were not essential for methylase activity except that the oligonucleotide substituted with inosine nucleotide at the first G position did not react with MspI methylase.
Kim
D S
DS
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Kang
Y K
YK
Yoo
O J
OJ
D001483:Base Sequence; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific