Characterization of the DNA-binding, regulatory, and methylating activities of the MTases Ecl18kI, SsoII, and their mutant forms1
| MTases | Relative yield of the RE gene transcript per unit of active concentration of MTase | Kd of the complex between MTase and the regulatory site, nM1,2 | Kd of the complex between MTase and the methylation site, nM1,3 | Relative initial methylation rate1,3 |
|---|---|---|---|---|
| Ecl18kI | 1.0 | 224 ± 24 | 87 ± 12 | 1 |
| SsoII | 1.0 | 248 ± 33 | 144 ± 14 | 1 |
| SsoII(C142A) | 1.0 | 35 ± 3 | 172 ± 10 | - |
| Ecl18kI(R15A) | 0.4 | 56 ± 13 | 103 ± 24 | < 1 |
| Ecl18kI(K21A) | 3.9 | 48 ± 9 | 87 ± 3 | 38 |
| cl18kI(K31A) | 1.0 | 198 ± 29 | 26 ± 3 | 29 |
| Ecl18kI(R35A) | - | > 4000 | 140 ± 12 | 2 |
| Ecl18kI(R38A) | - | > 4000 | 96 ± 13 | 11 |
| Ecl18kI(R39A) | 0.4 | 93 ± 14 | 266 ± 4 | 22 |
| Ecl18kI(R42A) | 2.5 | 32 ± 2 | 256 ± 4 | < 1 |
| Ecl18kI(K46A) | 13.5 | 250 ± 32 | > 4000 | - |
| Ecl18kI(K53A) | 1.8 | 206 ± 7 | > 4000 | - |
1 Data for M.Ecl18kl, M.SsoII, M.SsoII(C142A), M.Ecl18kl(R15A), M.Ecl18kl(R35A), M.Ecl18kl(R38A), M.Ecl18kl(R39A), and M.Ecl18kl(R42A) have been published earlier [9].
2 The complex formation was studied using the 31-bp DNA duplex IV containing the regulatory site: 5’-TTGGTTTTAGGACAATTTGTCCTGTTTTGAT-3’ 3’-AACCAAAATCCTGTTAAACAGGACAAAACGA-5’ (DNA duplex IV).
3 The complex formation and methylation activity were studied using the 30-bp DNA duplex V containing the methylation site: 5’-GATGCTGCCAACCTGGCTCTAGGTTCATAC-3’ 3’-CTACGACGGTTGGACCGAGATCGAAGTATG-5’ (DNA duplex V).