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. 2020 Dec 22;49(2):902–915. doi: 10.1093/nar/gkaa1224

Figure 6.

Figure 6.

Monoubiquitylation promotes SPRTN autocleavage in trans. (A) Recombinant SPRTN or a linear SPRTN-Ubiquitin fusion (SPRTN-UbLF) (500 nM) were incubated with histone H1 alone or in the presence of either single- (ss) Virion or double-stranded (ds) RFI ФX174 DNA (11.1 nM) for 60 min at 25°C. Salt concentrations were as indicated. Reactions were analysed by SDS-PAGE followed by western blotting and staining with InstantBlue Coomassie protein stain. Quantification of western blots of results of SPRTN and histone H1 cleavage: values represent the mean ± SD of four independent experiments. (B) Indicated model protein G-oligonucleotide conjugates (25nM) were incubated alone or in the presence of recombinant SPRTN (6.25 nM, WT or a linear SPRTN-Ubiquitin fusion (SPRTN-UbLF)) for 2 h at 25°C prior to separation by native PAGE. Right panel, quantification of DPC cleavage: values represent the mean ± SD of three independent experiments. (C) Recombinant catalytically inactive Flag-SPRTN-EQ (500 nM) was incubated alone or in combination with active SPRTN (500 nM, WT or a linear SPRTN-Ubiquitin fusion (SPRTN-UbLF)) in the presence of DNA (ФX174 RFI dsDNA, 11.1 nM) for 60 min at 25°C. Salt concentrations were as indicated. Reactions were subjected to SDS-PAGE followed by staining with InstantBlue Coomassie protein stain and western blotting.