Journal: Nucleic Acids Research

Article Title: Plasmid replication-associated single-strand-specific methyltransferases

doi: 10.1093/nar/gkaa1163

Figure Lengend Snippet: MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.

Article Snippet: All RE, DNA MTases, DNA substrates and markers, protein markers, and the PURExpress in vitro transcription-translation system were from New England Biolabs (NEB), MA.

Techniques: Activity Assay, Staining, Modification, In Vitro, Purification, Synthesized, In Vivo, Plasmid Preparation, Variant Assay, Lambda DNA Preparation

Journal: Nucleic Acids Research

Article Title: Plasmid replication-associated single-strand-specific methyltransferases

doi: 10.1093/nar/gkaa1163

Figure Lengend Snippet: Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.

Article Snippet: All RE, DNA MTases, DNA substrates and markers, protein markers, and the PURExpress in vitro transcription-translation system were from New England Biolabs (NEB), MA.

Techniques: Staining, Purification, Western Blot, Labeling, Activity Assay, Copurification, Sonication