Fig 2: TIMELESS Requires PARP1, but Not Its Activity, to Be Recruited to DNA Damage Sites(A) TIMELESS-Tomato and PARP1-GFP were stably co-transfected in U2OS cells and their recruitment to sites of DNA damage was captured by live cell imaging following laser microirradiation. Times are indicated in seconds.(B) U2OS cells stabling expressing GFP-TIMELESS were depleted of PARP1 for three days using siRNA oligo #1, and the kinetics of TIMELESS recruitment to DNA damage sites were assessed by live cell imaging of laser-induced lesions. For each condition, n = 28. SE is shown for each time point. Panels below show efficiency of knockdowns using western blot.(C) U2OS cells stabling expressing GFP-TIMELESS were treated for 1 hr with inhibitors to PARP1, ATM, DNA-PK, and CDK1 prior to laser microirradiation. Next, kinetics of TIMELESS recruitment to DNA damage sites were assessed by live-cell imaging of laser-induced lesions. PARP1 knockdown (using oligo #2) was used for comparison. For each condition, n = 20. SE is shown for each time point.(D) U2OS cells stabling expressing either wild-type GFP-TIMELESS or two GFP-TIMELESS mutants that do not bind PARP1 were subjected to laser micro-irradiation. Next, kinetics of recruitment of wild-type TIMELESS and TIMELESS mutants to DNA damage sites were assessed by live-cell imaging of laser-induced lesions. For each condition, n = 15. SE is shown for each time point.

Fig 3: Identification of the PARP1 Binding Site in TIMELESS(A) HEK293T cells were transfected with either an empty vector (EV), FLAG-tagged TIMELESS, or FLAG-tagged TIMELESS mutants as indicated. Asterisks indicate the insertion of a STOP codon after the numerically specified codon. 24 hr post-transfection, WCEs were subjected to IP with a-FLAG resin and immunoblotted as indicated.(B) Schematic representation of TIMELESS mutants tested for binding to PARP1. TIMELESS mutants that interacted with endogenous PARP1 are designated with the symbol (+); those unable to co-precipitate PARP1 are designated with the symbol (-).(C) The experiment was performed as in (A), except that different TIMELESS mutants were used.

Fig 4: TIMELESS Stabilizes PARP1 Interaction with Its Substrates upon DNA Damage(A) HEK293T cells were transfected with either an EV or the indicated FLAG-tagged proteins. 24 hr post-transfection, WCEs were subjected to IP with a-FLAG resin and immunoblotted as indicated. Ex. Prot., exogenous proteins.(B) The experiment was performed as in (A). Gels in the bottom panels were loaded as indicated in the upper panels, except that MWMs were omitted. Asterisks denote nonspecific bands.(C) U2OS cells were transfected overnight with either an siRNA to TIMELESS or a control non-targeting siRNA (siCTRL). 24 hr later cells were then transfected with either an EV or FLAG-tagged PARP1. 24 hr post-transfection, WCEs were subjected to IP with a-FLAG resin and immunoblotted as indicated. White asterisks denote non-specific band. The experiment was repeated three times with identical results.(D) PARP1 recruitment is impaired after laser microirradiation following knockdown of TIMELESS. U2OS cells stabling expressing PARP1-GFP were depleted of TIMELESS for three days using siRNA, and the kinetics of PARP1recruitment to DNA damage sites were assessed by live-cell imaging of laser-induced lesions. For each condition, n = 15. SE is shown for each time point.(E) KU80 recruitment is impaired after laser microirradiation following knockdown of TIMELESS. U2OS cells stabling expressing KU80-GFP were depleted of TIMELESS for 3 days using siRNA, and the kinetics of KU80 recruitment to DNA damage sites were assessed by live-cell imaging of laser-induced lesions. For each condition, n = 20. SE is shown for each time point.(F) Quantification of NHEJ measured as fold change in frequency of repair of EJ5-GFP, resulting in GFP-positive cells after expression of I-SceI in cells treated with the indicated siRNA oligos or drugs. Bar graphs represent the mean of eight independent experiments ± SD. p < 0.0001 (unpaired t test, using GraphPad software). Panel below shows efficiency of knockdown using western blot.(G) Quantification of HR measured as fold change in frequency of repair of DR-GFP, resulting in GFP-positive cells after expression of I-SceI in cells treated with the indicated siRNA oligos or drugs. Bar graphs represent the mean of eight independent experiments ± SD. p < 0.0001 (unpaired t test, using GraphPad software). Panels below show efficiency of knockdowns using western blot.