Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

Notes: This paper describes a potential "specificity checkpoint" for nucleotide incorporation by DNA polymerases. Using Tgo DNA polymerase from Thermococcus gorgonarius as a model system, the authors identified a single key residue (E664) that influenced the ability to incorporate rNTPs. Mutation of E664 to lysine (K) enabled RNA polymerization in the context of another mutation (the steric-gate mutation (Y409G)). The paper describes characterization of the mutant phenotype. As part of the study, luciferase mRNA was generated from the luciferase control DNA using the mutant polymerase. Synthesis and in vitro translation of the synthesized RNA was also evaluated using ssDNA templates, New England Biolabs PURExpress™ protein synthesis kit, and the FluoroTect™ Green_Lys in vitro translation labeling system. (4248)