TABLE 6.
Tetramer interface disrupting mutants found in AML and MDS patients show full activity but altered processivity
kcat and Km values were determined by monitoring the ability of the enzyme to incorporate tritiated methyl groups transferred from cofactor AdoMet onto DNA (poly-dIdC). koff values were determined by binding excess enzyme to 5′ FAM-6-labeled GCbox30 duplex and measuring the rate of change in anisotropy upon the addition of excess unlabeled GCbox30 (100-fold labeled DNA). Processivity was determined from the chase assay.
| kcat | Fold ↑ | KmAdoMet | Fold ↑ | KmDNA | Fold ↑ | koff | Fold ↑ | Processive | |
|---|---|---|---|---|---|---|---|---|---|
| h−1 | μm | μm bp | min−1 | ||||||
| WT | 3.5 ± 0.3 | 1.0 | 0.20 ± 0.02 | 1.0 | 1.2 ± 0.1 | 1.0 | 0.21 ± 0.01 | 1.0 | Yes |
| R771L | 6 ± 0.5 | 1.7 | 0.62 ± 0.09 | 3.1 | 16.3 ± 1.4 | 13.1 | 0.96 ± 0.05 | 4.5 | No |
| R729W | 2.9 ± 0.35 | 0.8 | 0.58 ± 0.09 | 2.8 | 8.4 ± 1.2 | 6.8 | 1.87 ± 0.20 | 8.8 | No |
| R736H | 0.68 ± 0.04 | 0.2 | 1.40 ± 0.40 | 6.9 | 21.5 ± 6.7 | 17.4 | 1.22 ± 0.10 | 5.8 | No |
| R882H | 1.41 ± 0.15 | 0.4 | 1.30 ± 0.14 | 6.4 | 6.6 ± 0.6 | 5.3 | 2.36 ± 0.04 | 11.1 | No |