More importantly and perhaps similar to dPds5, ORD seems not to be required for DSB repair

rrent model was deposited in the PDB after iterative geometry validation on the MOLPROBITY server,25 re-building with COOT26 and refinement with REFMAC.27 5.7. Selectivity assays Selectivity assays for buy MK 886 histone methyltransferases were performed as described by Siarheyeva et al.28 Selectivity experiments for compounds using different HMTs were performed by monitoring the incorporation of tritium-labeled methyl group to lysine or arginine residues of peptide substrates using scintillation proximity assay. After mixing, the reaction mixtures in Flashplate were incubated for 1 hour and the CPM counts were measured using Topcount plate reader. The CPM counts in the absence of compound for each data set were defined as 100% activity. The CPM counts in the absence of the enzyme in each data set were defined as background. IC50 values were calculated with the software SigmaPlot. For DNMT1, the IC50 determination was performed as described above by using dsDNA as a substrate. The dsDNA substrate was prepared by annealing two complementary strands, synthesized by Eurofins MWG Operon. DNMT1 was incubated with dsDNA substrate, 3H-SAM in buffer and the reactions were incubated for 1 hour at 37 C before quenching. Inhibition of methyltransferase activity of recombinant full-length human NNMT was measured using SAHH-coupled assay30. The reaction mixture consisted of 50 mM Tris-HCl, pH 7.5, 5 M SAM, 150 M nicotinamide, 5 M SAH hydrolase, 15 M ThioGlo 1, 0.5 M NNMT and variable amount of BrSAH ranging between 0.1 and 100 M. The methylation reaction was followed by measuring the increase in fluorescence using Synergy 4 microplate reader with 360/40 nm excitation filter and 528/20 nm emission filter for 30 min in 384-well plate format. Reaction rates were calculated from the initial linear slopes using Gen5 Software. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811292 Data was converted to percent activity, assuming 100% activity in the absence of BrSAH. IC50 values were calculated using a four parameter logistic equation using Sigma Plot software. Standard deviations were calculated from three independent experiments. 5.8. Chemistry All oxygen and/or moisture sensitive reactions were carried out under N2 atmosphere in glassware purged with N2 prior to use. All reagents and laboratory grade solvents were Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Bioorg Med Chem. Author manuscript; available in PMC 2016 March 07. Yu et al. Page 9 purchased from commercial vendors and used as received, without further purification. Nuclear Magnetic Resonance spectra were recorded on a Bruker Avance-III 500 MHz spectrometer. Proton chemical shifts are reported in ppm referenced to the NMR solvent; DMSO-d6 or D2O. Data are reported as follows: chemical shifts, multiplicity; coupling constant in Hz; integration; assignment. ~~ Mitosis ensures equal segregation of the genome to daughter cells, and defects in mitosis can lead to aneuploidy and polyploidy, frequently observed in cancers. Chromosome segregation requires assembly of the mitotic spindle and its attachment to sister kinetochores of all chromosomes, and is strictly controlled by the activity of the Spindle Assembly Checkpoint . Defects in kinetochore attachment prevent SAC satisfaction and chromosome segregation. Cells with compromised/defective SAC response may segregate their chromosomes erroneously and become aneuploid or polyploid. Aurora B kinase is one of the key mitotic factors controlling euploidy. Aurora B is a component of th