Fig 2: 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 3: Replisome components are required for interphase sister-chromatid cohesion.(A) Representative images of G2-enriched HeLa cells transfected with the indicated siRNAs and stained with DAPI (blue in merge) and the FISH probe (red in merge). Cells were treated with thymidine for 16–18 hr and released into fresh medium for 4 hr before fixation. Selected paired FISH signals are magnified in inset. Scale bar, 5 µm. (B) Quantification of the distances between paired FISH signals in G2-enriched HeLa cells transfected with the indicated siRNAs. Mean ± SD (siLuc, n = 502; siSororin, n = 243; siMCM2, n = 70; siCDC7, n = 44; siDBF4, n = 30; siDRF1, n = 30; siDBF4/DRF1, n = 32; siNIPBL, n = 191; siCDC45, n = 66; siGINS1, n = 68; siCDC45/GINS1, n = 26; siWDHD1, n = 198; siTIMELESS, n = 58; siTIPIN, n = 31; siTIMELESS/TIPIN, n = 51; siWDHD1/TIMELESS, n = 375; siWDHD1/TIMELESS/TIPIN, n = 71; siDDX11, n = 154; siWDHD1/DDX11, n = 52; siTIMELESS/DDX11, n = 65). The red dashed line indicates the mean of the siLuc sample. (C) Quantification of the percentage of cells with two unreplicated single FISH dots. Cells were transfected with the indicated siRNAs, treated with thymidine for 16–18 hr, and released into fresh medium for 4 hr. siLuc, n = 507 (mean ± SD; four independent experiments); siCDC7, n = 109; siNIPBL, n = 66; siCDC45, n = 71; siGINS1, n = 139; siCDC45/GINS1, n = 116; siFEN1, n = 112; siLIG1, n = 115; siCHAF1A, n = 226 (mean ± SD; two independent experiments). (D) Lysates of HeLa cells transfected with the indicated siRNAs were blotted with the indicated antibodies. (E) Quantification of the distances between paired FISH signals in G2-enriched HeLa cells transfected with the indicated siRNAs. Mean ± SD (siLuc, n = 382; siSororin, n = 82; siWDHD1/TIMELESS, n = 226; siFEN1, n = 60; siLIG1, n = 44; siCHAF1A, n = 110). (F) A simplified model delineating the molecular events during DNA replication. Cohesion establishment can occur without the maturation of Okazaki fragments and the deposition of histones.

Fig 4: TIMELESS Physically Interacts with PARP1(A) HEK293T cells were transfected with the indicated FLAG-tagged proteins. 24 hr post-transfection, cells were harvested and lysed. Cell extracts were subjected to immunoprecipitation with a-FLAG resin and stained with Ponceau S. MWM, molecular weight markers.(B) The experiment was performed as in (A), except that whole-cell extracts (WCEs) were subjected to immunoprecipitation (IP) with a-FLAG resin and immunoblotted as indicated. EV, empty vector. Asterisks denote FLAG-tagged proteins.(C) The experiment was performed as in (B).(D) Lysates of U2OS cells were immunoprecipitated with either an antibody against TIMELESS or a rabbit IgG and immunoblotted as indicated.(E) Super-resolution images of TIMELESS and PARP1 in U2OS cells selected from regions of the nucleus shown in Figure S1D. Scale bar, 400 nm.(F) Super-resolution images of TUNEL, TIMELESS, and PARP1 in neocarzinostatin treated U2OS cells showing that TIMELESS and PARP1 are found at DNA damage sites.

Fig 5: 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.