Figure 7.

Autocleavage Controls SPRTN Dynamics at Sites of DNA Damage

(A) SPRTN autocleavage occurs in trans. Recombinant GST-SPRTN-Strep WT and catalytically inactive YFP-E112Q-Strep were incubated in the absence or presence of ss and ds phage DNA (10 nM) for 2 hr at 25°C.

(B) SPRTN autocleavage occurs in cells. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells expressing the indicated SPRTN variants were lysed and subjected to SDS-PAGE followed by western blotting against the N-terminal YFP and the C-terminal Strep tag. The asterisk indicates an unspecific band serving as loading control.

(C) SPRTN autocleavage is triggered by formaldehyde. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells were treated with the indicated dose of formaldehyde for 2 hr.

(D) Schematic representation shows laser microirradiation and fluorescence recovery after photobleaching (FRAP) experiments.

(E) Recruitment of YFP-SPRTN-Strep (WT or EQ) in HeLa Flp-In TRex cells after laser microirradiation. Data are from ≥20 cells ± SEM normalized to pre-irradiation fluorescence.

(F) Representative images of HeLa Flp-In TRex cells expressing WT YFP-SPRTN-Strep from FRAP time course at indicated time following bleaching. Bleaching was achieved with 0.1-s pulse of 405-nM laser (scale bar, 10 μm).

(G) FRAP from HeLa Flp-In TRex cells expressing YFP-SPRTN-Strep (WT or EQ) data are from ≥15 cells ± SEM normalized to pre-bleach fluorescence (left panel). Fitted exponential fluorescence recovery of FRAP data is shown (right panel).