Fig. 2
A focused CRISPR-Cas9 mutagenesis “tag, mutate and enrich” screen identifies PARP1 mutations outside the DNA-binding domain that cause PARP inhibitor resistance. a Experimental “tag, mutate and enrich” scheme to isolate missense and in-frame PARP1 mutants associated with PARP inhibitor resistance. b Translated alignment of PARP1 amino-acid mutations identified in talazoparib-resistant cells isolated from lentiviral sgRNA pool 1 (designed to target ZnF1 and ZnF2 domains) from the screen shown in a. By comparing the multiple different mutations isolated from pool 1, a PARP1 p.119_120delKS minimal mutation associated with resistance was identified. Some insertions and larger deletions are omitted for clarity (see Supplementary Data 2). c PARP1 p.119_120delKS mutation abolishes recruitment of a PARP1-GFP fusion to sites of microirradiated DNA damage. CAL51 PARP1–/– cells were transfected with a wild-type PARP1-GFP cDNA construct or a PARP1 p.119_120delKS-GFP fusion cDNA expression construct and then exposed to localised ionising radiation (a microirradiated spot) as in f. Time course of PARP1-GFP signal at microirradiated site is shown. d Location of three mutations associated with PARPi resistance on a model of the PARP1 DNA structure12. e PARP1-N329Q and HD742F mutations ablate PARP1 trapping, while 848delY partially reduces trapping. Western blot from PARP1 trapping assay for three PARP1-GFP mutants and wild-type PARP1-GFP is shown. MMS-treated cells were lysed and fractionated into nuclear soluble (NS) and chromatin (C) fractions as described in Methods. Blot was probed with an anti-GFP antibody. f PARP1-848delY mutation alters PARP1 localisation kinetics at sites of DNA damage. Microirradiation and PAR synthesis phenotypes of N329Q and 848delY PARP1 mutants. Green—PARP1-GFP signal, red—PAR sensor (PBZ-mRuby2). Note lower recruitment relative to wild type of both mutants, but retention of PAR synthesis in 848delY. g Model of intramolecular communication between the DNA-binding ZnF1 domain and the catalytic (CAT) domain based on mutants identified from screens. (1) 45delD mutation in first zinc finger, (2) HD742F mutation shown in d, (3) E688 (Supplementary Data 2)
