XB-IMG-200401
Xenbase Image ID: 200401
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Fig. 1: DPCs on ssDNA gaps are ubiquitylated in a PARP1-dependent manner.
A Schematic of DPC pull-down assay9. At given time points, the DPC plasmid is pulled down under stringent conditions, the DNA is digested by benzonase treatment, and M.HpaII is analyzed via immunoblotting. Note that although the M.HpaII antibody is generated against full-length M.HpaII, it is unlikely to recognize all degradation products. B pMHssDNA or pMHdsDNA were incubated in high-speed supernatant extract (HSS, which is an extract that does not support DNA replication) and recovered by DPC pull-down at the indicated time points and immunoblotted for crosslinked M.HpaII. Molecular weight marker (kDa) is indicated on the left side of the blot and in all subsequent blots presented in this manuscript. C pMHssDNA was incubated in SPRTN- or SPRTN- and RFWD3-depleted HSS and retrieved at the indicated time points by DPC pull-down as in B. D pMHssDNA repair in SPRTN- and RFWD3-depleted HSS. Reactions were supplemented with untagged- or FLAG-tagged recombinant ubiquitin. For each condition, a sample was retrieved at 1 and 10 min, and was first recovered by DPC pull-down (DPC-PD), subsequently by FLAG pull-down (FLAG-PD), and immunoblotted for crosslinked M.HpaII. E pMHssDNA was incubated in SPRTN- and RFWD3-depleted HSS in the presence of the indicated inhibitors. DPCs were recovered via DPC pull-downs at the indicated time points and immunoblotted for crosslinked M.HpaII. Note that the small upshift of M.HpaII signal observed in the presence of Ub.E1i (lanes 5 and 6) is due to M.HpaII PARylation (see Fig. 2D). F pMHssDNA repair in SPRTN- and RFWD3-depleted HSS, which was further mock- or PARP1-depleted in the presence and absence of PARP inhibitor (PARPi). Samples were recovered by DPC pull-down and immunoblotted for crosslinked M.HpaII. The asterisk marks an unspecific band. G Scheme of PARP1. It consists of three main domains: an N-terminal DNA-binding domain (DBD) consisting of zinc-finger (ZF) motifs, a central BRCT domain-containing automodification domain, and a conserved C-terminal catalytic domain (CD). H SPRTN-RFWD3-depleted HSS was further mock- or PARP1-depleted and blotted with PARP1 antibody. PARP1-depleted extracts were supplemented with either buffer (+Buf.), recombinant hPARP1 (+WT), or recombinant catalytically impaired E988K mutant (+E988K). I Add-back rescue experiment using the extracts from H. Samples were recovered by DPC pull-down and immunoblotted against M.HpaII. Source data are provided as a Source Data file. Image published in: Fábián Z et al. (2024) © The Author(s) 2024. Creative Commons Attribution-NonCommercial-NoDerivatives license Larger Image Printer Friendly View |