Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 is required for replication stress response. ( A, C, E ) Survival assays of control and DOCK7-depleted U2OS cells treated with indicated doses of IR, HU or CPT. Data are represented as the mean ± SEM of n = 3 independent experiments. ( B, D, F ) Phosphorylation of CHK1 and CHK2 were determined by immunoblotting in control and DOCK7-depleted U2OS cells treated with 10 Gy IR, 10 mM HU or 1 μM CPT for 2 h. ( G-J ) U2OS cells were labeled with 50 μM IdU, and then treated with or without HU, thereafter incubated with 200 μM CldU for indicated time, the fork speed and the length of CIdU track in control and DOCK7-depleted cells were determined by measuring the length of CIdU track panels (H and J), representative pictures of fibers are shown in panels (G and I). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001. ( K ) ER-AsiSI U2OS cells were transfected with vector control or FLAG-DOCK7 for 36 h before being treated with or without 1 μM 4-OHT for 4 h. After cells were harvested, ChIP experiments were performed using FLAG antibody. Error bars represent SEM from three independent experiments. ***p<0.001.

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: Western Blot, Labeling, Incubation, Two Tailed Test, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 is phosphorylated by ATR and then recruited to chromatin by MDC1 to regulate replication stress response. ( A and H ) HEK293T cells stably transfected with HA-MDC1 (A), FLAG-DOCK7 WT or S1438A mutant (H) were incubated with 10 μM EdU for 20 min before treated or untreated with HU. Replication fork recruited proteins were isolated by iPOND and blotted with indicated antibodies. ( B and E ) Control and MDC1-depleted or ATR-depleted U2OS cells were treated with 10 mM HU for 1 h, cells were then harvested and separated into chromatin and soluble fractions, the protein level of DOCK7 in each fraction was detected by immunoblotting assay. ( C ) HEK293T cells were transfected with indicated MDC1 constructs for 24 h, co-immunoprecipitation (co-IP) assay were performed using anti-HA agarose beads and then blotted with indicated antibodies. ( D ) HEK293T cells transfected with FLAG-DOCK7 were pre-treated with DMSO or 50 nM VX-970 for 2 h then treated 10 mM HU for 2 h, cell lysates were immunoprecipitated with anti-FLAG agarose beads, and left untreated or were treated with phosphatase, cell lysates were blotted with the indicated antibodies. ( F ) HEK293T cells transfected with WT or S1438A mutant of DOCK7 were incubated with 10 mM HU for 1 h, cell lysates were then immunoprecipitated with anti-FLAG agarose beads and blotted with indicated antibodies. ( G ) The protein levels of FLAG-DOCK7 in chromatin and soluble fractions of HEK293T cells transfected with WT or S1438A mutant of FLAG-DOCK7 before or after HU treatment were detected by immunoblotting assay. ( I ) Lysates from HEK293T cells transfected with WT or S1438A mutant of DOCK7 with or without HU treatment were used in a PAK-CRIB pull-down assay. The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. ( J ) The survival rate of control or DOCK7-depleted U2OS cells transfected vector control or indicated DOCK7 constructs were assessed by colony formation assay. Error bars represent SEM from three independent experiments.

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: Stable Transfection, Transfection, Mutagenesis, Incubation, Isolation, Western Blot, Construct, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Plasmid Preparation, Colony Assay

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 increases the protein stability of RPA1 in chromatin and replication fork. ( A ) The distribution of indicated proteins in the chromatin and soluble fractions of control or DOCK7-depleted U2OS cells after treated with 10 mM HU for 2 h were determined by immunoblotting assay. ( B and C ) Representative images (B) and quantification (C) of RPA2 foci. More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( D ) Control and DOCK7-depleted HEK293T cells were incubated with 10 μM EdU for 20 min before or after HU treatment. Replication fork recruited proteins were isolated by iPOND and blotted with indicated antibodies. ( E ) Chromatin and soluble fraction of cell lysates separated from MG132-treated HEK293T were blotted to measure the expression level of RPA1. ( F ) The protein contents of RPA1 in the chromatin and soluble fraction of control or DOCK7-depleted HEK293T cells treated with 20 μM CHX for different time points were detected by immunoblotting assay. ( G ) Control or DOCK7-depleted HEK293T cells were transfected with FLAG-RPA1 and His-Ub for 24 h before being treated with 10 mM HU for 1 h, the chromatin and soluble fractions of harvested cell lysates were then immunoprecipitated with nickel (His) beads and blots were probed with indicated antibodies.

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: Western Blot, Incubation, Isolation, Expressing, Transfection, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7-Rac1/Cdc42-PAK1 pathway is critical for replication stress response through regulating the chromatin recruitment of RPA1. ( A-C ) Control or DOCK7-depleted U2OS cells were transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 truncates for 24 h before treated with 10 mM HU for 1 h. The expression levels of RPA1 and RPA2 in the chromatin and soluble fraction of harvested cells were determined by immunoblotting assay (A); RPA2 foci formation was detected by immunofluorescence (B) and quantified (C). More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( D ) Survival assays of control or DOCK7-depleted U2OS cells transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 in response to HU. Error bars represent SEM from three independent experiments. ( E ) HEK293T cells were transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 for 24 h before treatment with 10 mM HU for 2 h, cells lysates were then used in a GST-PAK-CRIB pull-down assay to detect the activation of Rac1 and Cdc42. ( F and I ) Chromatin and soluble fractions of cell lysates derived from DMSO or 1 μM R-ketorolac pretreatment (F) and control or PAK1 knockdown (I) U2OS cells were subjected to immunoblot with the indicated antibodies. ( G, H , J and K ) Representative images (G and J) and quantification (H and K) of RPA2 foci in DMSO or R-ketorolac pretreatment (G and H) and control or PAK1 knockdown (J and K) U2OS cells after HU treatment were detected and analyzed by immunofluorescence. ( L ) RPA1 and RPA2 proteins isolated by iPOND from control or PAK1 depletion HEK293T cells were detected by immunoblotting assay. ( M and N ) DNA fiber assay was performed to detect the length of CIdU track after HU was removed in control or PAK1-depleted U2OS cells (N), representative pictures of fibers are shown in (M). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001.

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Pull Down Assay, Activation Assay, Derivative Assay, Isolation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: RPA1 phosphorylated by PAK1 at Ser-135 and Thr-180 is critical for its role in replication stress response. ( A–C ) Control or DOCK7-depleted HEK293T cells were transfected with indicated FLAG-RPA1 constructs for 24 h. Cells were then pretreated with or without inhibitors (R-ketorolac or PAK1 inhibitor) before or after HU treatment. FLAG-RPA1 was coimmunoprecipitated from cell lysates and loaded on both normal and Phospho-tag gel, thereafter blotted with indicated antibodies. ( D ) Purified WT and ST/A mutant of RPA1 were incubated with or without constitutively active PAK1 (50 aa-150 aa) and incubated with γ- [32P] ATP for 30 min at 30°C before subjected to autoradiography. ( E–G ) Control or RPA1-depleted U2OS cells were transfected with WT or ST/A mutant of FLAG-RPA1 for 24 h before treatment with 10 mM HU for 1 h, and then the distribution of RPA1 and RPA2 in the chromatin and soluble fractions of cells were determined by immunoblotting (E). Representative images (F) and quantification (G) of RPA2 foci were analyzed by immunofluorescence. More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( H–J ) RPA1-depleted cells were transfected with WT or ST/A mutant of FLAG-RPA1 for 24 h before HU treatment, and then IPOND assay in HEK293T cells was performed to detect the distribution of the WT or ST/A mutant of RPA1 on the replication fork (H). DNA fiber assay in U2OS cells was performed to detect the length of CIdU track after HU was removed (J), and the representative pictures are shown in panel (I). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001. ( K ) Control or RPA1-depleted U2OS cells were transfected with vector control, WT or ST/A mutant of FLAG-RPA1 for 24 h, treated with the indicated concentrations of HU and survival was measured using the colony formation assay. Error bars represent SEM from three independent experiments. ( L ) The protein stability of WT or ST/A mutant of RPA1 in the chromatin and soluble fraction of RPA1-depleted HEK293T cells under CHX treatment were analyzed by immunoblotting assay. ( M ) RPA1-depleted HEK293T cells were transfected with WT or ST/A mutant of FLAG-RPA1 and His-Ub for 24 h before HU treatment. Chromatin and soluble fractions derived from harvested cells were immunoprecipitated with nickel (His) beads and then blotted with indicated antibodies

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: Transfection, Construct, Purification, Mutagenesis, Incubation, Autoradiography, Western Blot, Immunofluorescence, Two Tailed Test, Plasmid Preparation, Colony Assay, Derivative Assay, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 depletion enhances chemotherapy of ovarian cancer cells in vitro and in vivo . ( A ) Cell survival rate after CPT treatment was determined by colony formation assay in control or DOCK7-depleted OVCAR8 cells. Error bars represent SEM from three independent experiments. ( B–D ) Control or DOCK7-depleted OVCAR8 cells were subcutaneously injected into the flank of NOD-SCID mice. Mice were treated with saline or CPT (10 mg/kg i.p. 3 days × 8 times). Tumor images were shown in (B), and tumor weight (C) and volume (D) were assessed. Data points (mean ± SEM) are shown from five biologically independent samples by two-sided unpaired t test. ( E ) Working model of RPA1 phosphorylation regulated by DOCK7 signaling. DOCK7 is phosphorylated by ATR during replication stress and then recruited to the chromatin and DNA damage sites by MDC1. Thereafter, DOCK7 facilitates the GTP-loading of Rac1/Cdc42, which in turn activate PAK1 to phosphorylate and stabilize chromatin-loaded RPA1 to stabilize and enable replication fork restart.

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: In Vitro, In Vivo, Colony Assay, Injection