Journal: Nucleic Acids Research
Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI
Figure Lengend Snippet: TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.
Article Snippet: Plasmid based assays TagI restriction activity was first assessed by digestion of 0.5–1 μg of pBR322 (Dcm+ or Dcm− ), pBRFM+ (Dcm+ ) or pACYC-HpyCH4IVM+ plasmid in NEB buffer 2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9) at 37°C for 1 h. After the reaction Protease K (1.6 U) was added at 37°C for 15 min.
Techniques: Activity Assay, Polymerase Chain Reaction, Modification, In Vitro, In Vivo, Lysis, Infection, Expressing