Fla 5000

DNA oligonucleotides used for nuclease activity assays were synthesized (BEX Co., Tokyo, Japan or MDBio, Inc., Taiwan) and labeled at the 5′ end with [γ-³²P]ATP by T4 polynucleotide kinase and purified on a Microspin G-25 column (GE Healthcare) to remove the nonincorporated nucleotides. Purified substrates (20 nM; see Table S1 for sequences) were incubated with RNase T, ExoI, or ExoX at various concentrations in a buffered solution of 120 mM NaCl, 2 mM MgCl₂, and 50 mM Tris-HCl (pH 7.0) at room temperature for 20–60 min. The reaction was quenched by addition of the stop solution (2× TBE) and heating at 95°C for 5 min. Reaction samples were then resolved on 20% denaturing polyacrylamide gels and visualized by autoradiography (Fujifilm, FLA-5000).
DNA binding affinities of RNase T, ExoI, and ExoX were measured by gel shift assays. The 5′-end ³²P-labeled DNA substrates (20 nM) were incubated with RNase T, ExoI, or ExoX in a solution of 100 mM NaCl, 30 mM EDTA, 10 mM EGTA, and 50 mM Tris-HCl (pH 7.0) for 20 min at room temperature. The concentrations of each protein used in the assays were 0, 5, and 50 µM. Reaction samples were then resolved on 20% TBE gels (Invitrogen) and visualized by autoradiography (Fujifilm, FLA-5000).

Kinetic of 3′-P and ST activities were performed as previously described^{21 (link)}. Briefly, WT IN or mutant proteins (400 nM) were mixed with 20 nM radiolabeled DNA substrates in a buffer containing 50 mM MOPS pH 7.2, 7.5 mM MgCl₂, 14 mM 2-mercaptoethanol at 37 °C. Reactions were stopped after 10, 20, 30, 45, 60, 90 or 120 minutes by addition of an equal volume of loading buffer (formamide containing 1% SDS, 0.25% bromophenol blue, and xylene cyanol). Reaction products were separated in 16% poly-acrylamide denaturing sequencing gels. Image of the gels was obtained after autoradiography using a FLA-5000 (Fujifilm). Densitometric analyses were performed using ImageQuant 5.1 software from GE Healthcare.