Journal: Nucleic Acids Research

Article Title: Structure-functional characterization of Lactococcus AbiA phage defense system

doi: 10.1093/nar/gkae230

Figure Lengend Snippet: DNA polymerization assay for AbiA wild type protein and its variants (radioactivity readout). ( A ) Template-independent polymerization activity of AbiA variants. Protein (0.5 μM) was incubated with 5 μCi α- 33 P-dATP (concentration 0.16 μM) at 37°C for 1 minute in the reaction buffer (50 mM Tris [pH 7.0], 250 mM NaCl, 10 mM MgCl 2 , and 5 mM DTT), to label primers copurified with protein. After labelling, half of the reaction was diluted fourfold in the reaction buffer containing an equimolar mix of 150 μM of all four deoxynucleotide triphosphates and aliquots of primer extension reactions were collected to a stop mix containing EDTA and proteinase K. Reaction products were analyzed on 15% TBE-urea polyacrylamide gels and visualized by autoradiography. Each set of four reactions consists of a primer labelling reaction (diluted four-fold in water, lane ‘0’) and primer extension time points (1.5, 3 and 4.5 min—black triangle). M – Single-stranded DNA marker made of 5′end-labeled oligonucleotides. ( B ) Analysis of labeled nucleotides covalently attached to AbiA variants. Protein (0.5 μM) was incubated with α- 33 P-dATP (0.16 μM) at 37°C in the presence of reaction buffer for 30 min. Protein samples were analyzed on a 12% SDS-PAGE gel and visualized by autoradiography (lower panel), then stained with Coomassie Blue (upper panel). M, protein size marker. ( C ) Deoxynucleotide preference of template-independent polymerization activity of AbiA. Aliquots of primer labelling reaction of wt AbiA (prepared as described in A) were diluted twofold in four extension mixes containing the reaction buffer with 200 μM of dGTP, dATP, dTTP or dCTP. Primer extensions were stopped after 5 min. Reaction products were analyzed on 15% TBE-urea polyacrylamide gels and visualized by autoradiography. M – Single-stranded DNA marker made of 5′end-labeled oligonucleotides. ( D ) Template-independent polymerization activity of SUMO-AbiA variants. For description see (A).

Article Snippet: For the biochemical experiments, we used α- 33 P-dATP 3000 Ci/mmol, 10 mCi/ml, 250 uCi, Hartman Analytics.

Techniques: Polymerization Assay, Radioactivity, Activity Assay, Incubation, Concentration Assay, Autoradiography, Marker, Labeling, SDS Page, Staining