Fig. 2: PARP1-dependent DPC PARylation triggers DPC ubiquitylation and resolution.

A Overview table of PARP1 mutants. B SPRTN-RFWD3-depleted HSS was further mock- or PARP1-depleted and immunoblotted with PARP1 antibody. PARP1-depleted extracts were also supplemented with either buffer (+Buf.), DNA-binding deficient mutant (+ΔZF1-2), automodification-deficient mutant (+3SA), or catalytically impaired mutant PARP1 (+E988K). C Add-back rescue experiment using the extracts from B. DPCs were recovered by DPC pull-down and monitored via blotting against M.HpaII. The asterisk denotes an unspecific band. D pMH^ssDNA was incubated in RFWD3-SPRTN-depleted HSS in the presence of PARPi or E1 ubiquitin-activating enzyme inhibitor (Ub.E1i) alone or in combination. Samples were recovered via DPC pull-down and immunoblotted with either M.HpaII or Poly/Mono-ADP Ribose antibody (α-PAR). E pMH^ssDNA was incubated in SPRTN-RFWD3-depleted HSS, which was then either mock- or PARP1-depleted in the presence of Ub.E1i where indicated. DPC degradation and PARylation were monitored as in D. F pMH^ssDNA and pMH^dsDNA were incubated in SPRTN-RFWD3-depleted HSS supplemented with PARGi where indicated. DPC degradation and PARylation were monitored as in D. Source data are provided as a Source Data file.