Project description:DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
Project description:Previous study revealed that the protective effect of TIGAR in cell survival is mediated through the increase in PPP (pentose phosphate pathway) flux. However, it remains unexplored if TIGAR plays an important role in DNA damage and repair. This study investigated the role of TIGAR in DNA damage response (DDR) induced by genotoxic drugs and hypoxia in tumor cells. Results showed that TIGAR was increased and relocated to the nucleus after epirubicin or hypoxia treatment in cancer cells. Knockdown of TIGAR exacerbated DNA damage and the effects were partly reversed by the supplementation of PPP products NADPH, ribose, or the ROS scavenger NAC. Further studies with pharmacological and genetic approaches revealed that TIGAR regulated the phosphorylation of ATM, a key protein in DDR, through Cdk5. The Cdk5-AMT signal pathway involved in regulation of DDR by TIGAR defines a new role of TIGAR in cancer cell survival and it suggests that TIGAR may be a therapeutic target for cancers.
Project description:Treatment with the histone deacetylase inhibitor trichostatin a (TSA) changes the radial positioning of the CFTR gene in HeLa S3 cells. The gene relocates from the nuclear periphery to the nuclear interior. In Calu-3 cells the gene is located in the nuclear interior. To identifiy potential regulatory elements for the positioning of CFTR, the histone h3 and h4 acetylation patterns of untreated and TSA-treated HeLa S3 and untreated Calu-3 cells were determined by ChIP-chip. A CTCF site close to the CFTR promoter displayed consistent histone H3 hyperacetylation in TSA treated HeLa S3 cells and Calu-3 cells.
Project description:The distribution of histone variants H2Abbd and macroH2A in 13 regions of the HG18 assembly have been studied using a variant of the ChIP-on-Chip technique. HeLa S3 cell lines expressing tagged histones H2A, H2Abbd or macroHA were obtained using retroviral transfer. DNA fractions associated with tagged histones were isolated using a two-step purification procedure that involved affinity chromatography on a column with anti-FLAG antibodies, followed by affinity chromatography on a Ni-agarose column. The obtained genomic DNA samples were analyzed by hybridization with custom NimbleGene genomic microarrays. Two samples. Test sample 1 is HeLa S3 cells expressing epitope-tagged histone H2Abbd and test sample 2 is HeLa S3 cells expressing epitope-tagged histone macroH2A . The control for both test sample 1 and test sample 2 is HeLa S3 expressing epitope-tagged histone H2A. Two copies of each probe per array were made.
Project description:Bcl2 can enhance susceptibility to carcinogenesis, but the mechanism(s) remains fragmentary. Here we discovered that Bcl2 suppresses DNA double-strand-break (DSB) repair and V(D)J recombination by downregulating Ku DNA binding activity, which is associated with increased genetic instability. Exposure of cells to ionizing radiation enhances Bcl2 expression in the nucleus, which interacts with both Ku70 and Ku86 via its BH1 and BH4 domains. Removal of the BH1 or BH4 domain abrogates the inhibitory effect of Bcl2 on Ku DNA binding, DNA-PK, and DNA end-joining activities, which results in the failure of Bcl2 to block DSB repair as well as V(D)J recombination. Intriguingly, Bcl2 directly disrupts the Ku/DNA-PKcs complex in vivo and in vitro. Thus, Bcl2 suppression of the general DSB repair and V(D)J recombination may occur in a mechanism by inhibiting the nonhomologous end-joining pathway, which may lead to an accumulation of DNA damage and genetic instability.
Project description:Identification of 2′–5′ OA binding proteins in human (HeLa), mouse (BMDM) and fly (Schneider S2) cells using affinity purification mass spectrometry (AP-MS). Pulsed SILAC-based translational profiling in HeLa and HeLa S3 cells treated with 2′–5′ OA or OH-2′–5′ OA.
Project description:We determined the occupancy of SUMO-1 on chromatin in HeLa cells by use of chromatin affinity purification coupled with next generation sequencing 18 samples examined in syncrhonized HeLa cells: Cells in G1, early S (S0)/mid (S3)/ late S phase (S6), and mitosis (M), triplicates for each sample. 1 ChIP-SUMO1 sample in S0, and 1 ChIP-IgG control
Project description:Me3K27 Histone H3 ChIP DNA was isolated from HeLa S3 cells and surveyed for binding in the ENCODE regions on NimbleGen ENCODE arrays. Use of this data requires permission from its producers. Keywords: ChIP-chip
Project description:Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6-4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6-4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 ?s time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.
Project description:The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base-base and insertion-deletion mismatches generated during DNA replication. To systematically investigate the mismatch repair pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using a tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. In total, we identified 262 high-confidence candidate interaction proteins (HCIPs).