Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Phosphorus-containing analogues of methyltransferase substrates/inhibitors and selected reactions catalyzed by these enzymes. ( a ) Structures of the H-phosphinic analogue of methionine (L-Met-P H ), (S,S)-SAM, H -phosphinic analogues of SAM and SAH ((R,S)-SAM-P H and rac-SAH-P H ), and phosphonic analogues of SAM and SAH (rac-SAM-P 5 and rac-SAH-P 5 ). H-Phosphinic and phosphonic groups are marked in yellow and rose, respectively. Individual diastereomers have hashed wedge bonds at the chiral centres, while the corresponding racemates are depicted using solid bonds. ( b ) DNA-(cytosine C5) methylation at CpG sites catalyzed by Dnmt3a, which establishes de novo DNA methylation patterns , and methylation at hemimethylated CpG sites catalyzed by Dnmt1, which maintains DNA methylation patterns during replication in mammals . ( c ) Methylation of protocatechuic aldehyde catalyzed by catechol-O-methyltransferase (COMT).

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Analogues, Methylation, DNA Methylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Effect of SAM phosphorus-containing analogues on Dnmt1 activity. ( a ) Methylation of D1 hairpin DNA fragment by Dnmt1 in a time-dependent and SAM concentration-dependent manner at 37 °C for 4–24 h using 1.0 µM and 5.0 µM SAM. ( b ) Dnmt1 methylation activity (24 h) in the presence of SAM phosphorus-containing analogues: rac-SAM-P H (100 µM), (R,S)-SAM-P H (20 µM and 50 µM), or rac-SAM-P 5 (20 µM and 100 µM), compared with SAM (5 µM). In both panels, the control was D2 hairpin DNA (a product of methylation) digested with GlaI. In both panels the results indicate means from five independent experiments. Error bars represent standard deviations. ** p ≤ 0.01 according to one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Analogues, Activity Assay, Methylation, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Interaction of SAM and SAH phosphorus-containing analogues with Dnmt1 and COMT. ( a ) Inhibition of Dnmt1 methylation activity. Dnmt1 reaction mixtures contained the following: SAM (5 µM), rac-SAH-P 5 (100 µM), rac-SAM-P 5 (100 µM), rac-SAH-P H (100 µM), or (R,S)-SAM-P H (100 µM). SAH (25 µM) was used as a reference inhibitor. The D2 DNA hairpin, the product of the Dnmt1 reaction cleaved with GlaI, was used as the control. ( b ) Inhibition of COMT methylation activity. Reaction mixtures contained either SAM or (R,S)-SAM-P H (each 6 mM) as methyl group donors and SAH (3 mM) or rac-SAH-P H (3 mM) as inhibitors. The duration of the reactions was 2 h and 24 h. In both panels the results indicate means from five independent experiments. Error bars represent standard deviations. ** p ≤ 0.01 according to one-way ANOVA with Dunnett’s post hoc test (vs. “no inhibitor”).

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Analogues, Inhibition, Methylation, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Dnmt1 activity assay using a hemimethylated DNA hairpin D1 and the endonuclease GlaI, making a double-strand break into the fully methylated hairpin D2 that results in enhanced fluorescence. The methylated CpG site is located within the endonuclease GlaI recognition site (GCGm 5 C/Gm 5 CGm 5 C).

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Activity Assay, Methylation, Fluorescence