Enzymatic methylation assays are performed on full-length, truncated and mutant DNMT1 using 14-nucleotide oligomer DNA duplex containing a single CpG dinucleotide in either hemi-methylated (measure maintenance activity) or un-methylated state (measure de novo activity). Rates of 3H-CH3 in nMol per hour transferred are measured against CG sites in μM.
The 4 methylases tested are mDNMT1 (650-1602), mDNMT1 K686A/Q687A (650-1602) with two mutations in CXXC domain, truncated mDNMT1(717-1602) and mDNMT1 C1229S(650-1602) with a mutation at catalytic center. The 14-mer DNA duplex is used as substrates.
(A) Domain structures of mouse DNMT1 proteins used for activity assay. The Lys686Ala/Gln687Ala
and Cys1229Sermutations are shown as yellow bars. (B) Sequence of 14-nucleotide oligomer DNA duplex used for enzymatic assay.
and Cys1229Sermutations are shown as yellow bars. (B) Sequence of 14-nucleotide oligomer DNA duplex used for enzymatic assay.
The enzymatic data is shown below,
(C) Enzyme kinetics on de novo and maintenance methylation of DNMT1 mutants after removal or mutation of CXXC domain. Rates of 3H-CH3 in nMol per hour transferred to unmethylated and hemimethylated DNA duplexes by 1 nM of mDNMT1(650–1602) wild-type, Lys686Ala/Gln687Ala and Cys1229Ser mutants and mDNMT1(717–1602) proteins were plotted as a function of CpG site concentration; the steady-state Michaelis-Menten parameters that were estimated from these plots are listed in table S3. Each reaction point was repeated in triplicate; mean and SD values are shown.
mDNMT1(717-1602) is the truncated enzyme with CXXC and a segment of CXXC-BAH1 linker deleted. On un-methylated substrate, it has a kcat increased from 0.60±0.03 hour-1 for the 650–1602 protein to 3.3±0.2 hour–1 for 717–1602 protein. In contrast, there is a small drop in kcat on hemi-methylated DNA from 650–1602 (kcat = 45±6 hour−1) to 717–1602 (kcat=36±3hour−1) mDNMT1.
Also, it has been found that full-length hDNMT1(1-1616), the initial rate kcat for un-methylated and hemi-methylated substrate is 0.25±0.04 and 18±2 hour-1 whereas the data for truncated hDNMT(646-1600) is 0.18±0.02 and 12±1 hour-1.
In addition, the mutant mDNMT1 K686A/Q687A (650-1602) confirmed by gel shit assay has a mutation in the residues that have contact with G within the CpG site which interrupt the contact. This mutated form also has anincrease in kcat (2.1±0.2 hour−1) on un-methylated substrates and a modest decrease in kcat (22±2 hour−1) on hemi-methylated substrates compared with full-length mDNMT1.
In conclusion, all the data suggests that the enzyme activity has a preference of hemi-methylated DNA on full-length hDNMT1 corresponding to the structural data of auto-inhibition for un-methylated DNA. The preference is mainly a result from the CXXC and auto-inhibitory linker since either removal or mutagenesis of the CXXC or auto-inhibitory domain increases the catalytic activity of mDNMT1 on un-methylated DNA.
All the figures used in this post are all from Jikui Song, et al. Science 331, 1036 (2011)
