Figure 4. In vitro SPRTN substrate cleavage.
(A) In vitro SPRTN self-cleavage reactions. Purified proteins were incubated with or without DNA for 5 hr at 37°C. 1,10 Phe = 1,10 Phenanthroline, a zinc metal chelator and inhibitor of zinc metalloproteases. Proteins were separated by SDS-PAGE and stained with coommasie blue. (B) In vitro histone H3 cleavage. SPRTN was incubated with or without histone H3 (SPRTN:H3 molar ratio of 4:1) in the presence of dsDNA for the indicated time points. SPRTN-E112A or SPRTN-Y117C mutants were incubated for 2 hr. Proteins were separated on an SDS-PAGE gel and transferred to a membrane for Western blot analysis. Histone H3 cleavage as well as SPRTN self-cleavage were monitored by immunobloting with antibodies against histone H3 and SPRTN. (C) In vitro Top2 cleavage. Purified recombinant Top2 was pre-incubated with DNA and Etoposide to irreversibly bind Top2 to DNA. Recombinant SPRTN or SPRTN-E112A was then added alone or in combination with 10-fold molar excess of either ubiquitin or SUMO and incubated for 2 hr at 37°C. Proteins were separated by SDS-PAGE and transferred to a membrane for Western blot analysis. Membrane was stained with amido black to detect SPRTN cleavage fragments.
Figure 4—figure supplement 1. SPRTN recruitment to chromatin.
Figure 4—figure supplement 2. SPRTN activation by ubiquitin.
