Extended Data Fig. 2: HPF1 recruitment to sites of damage relies on interaction with the C-terminus of PARP1.

(a, b) Western blot analysis (a) and representative confocal images (b) of YFP-HPF1 expression levels in U2OS Flp-In/T-Rex HPF1^KOcells after doxycycline induced expression. Transient transfection with YFP-HPF1 was also used to further boost HPF1 expression in the presence of 25 ng/mL Doxycycline. Images were taken 30 s post 405-nm laser irradiation. Scale bar, 10 μm. (c) Quantification of the mean nuclear fluorescence intensity of cells from b. Fluorescence level is normalised to 5 ng/mL (endogenous levels of HPF1 expression according to a). (d, e) Mean (d) or normalized (e) recruitment kinetics of YFP-HPF1 after 405-nm laser irradiation in U2OS Flp-In/T-Rex HPF1^KOcells with different levels of doxycycline induction. Individual recruitment curves from d were normalised to the peak HPF1 recruitment levels. Data from b-e are a representative of 2 independent replicates where data were collected from 12–18 cells per condition. (f) Recruitment kinetics of GFP-HPF1 in PARP1^KO or PARP1/HPF1 double knockout cells expressing mCherry-PARP1 WT. Data from f are a representative of 2 independent replicates where data were collected from 10–12 cells per condition. (g) Immunoblots of whole-cell extract from U2OS WT, PARP1^KO, HPF1^KO and PARP1/HPF1 double knockout cells. (h) Recruitment kinetics of GFP-HPF1 to sites of DNA damage induced by 405-nm laser irradiation in WT or PARP1^KO cells expressing WT N-terminally (mCh-PARP1) and C-terminally tagged PARP1 (PARP1-mCh). Data from h are a representative of 3 independent replicates where data were collected from 12–16 cells per condition. (i) Schematic representation of PAR-3H assay. (j, k) Representative confocal images (j) or quantification (k) of mCherry tagged PARP1 WT, PARP1 3SA or PARP1 E988K recruitment to YFP tagged macrodomain of mH2A1.1 tethered to LacO, Pre or 30 s post-irradiation. Inset, pseudocolored according to the look-up table displayed, shows the magnified LacO array. Scale bar, 5 µm. Data from k are a representative of 3 independent replicates where data were collected from 13–15 cells per condition. (l, m) Normalised recruitment kinetics of mCherry tagged PARP1 (WT, 3SA or E988K) (l) and GFP-HPF1 (m) expressed in PARP1^KO cells from Fig. 1f–h. Data from l-m are a representative of 3 independent replicates where data were collected from 13–18 cells per condition.

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