DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci.

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that have low levels of pre-existing hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.

Novel Pt(IV) Prodrugs Displaying Antimitochondrial Effects.

The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of cis,cis,trans-[Pt(NH3)2Cl2(L3)(OH)] (C-Pt1), cis,cis,trans-[Pt(NH3)2Cl2(L3)(BZ)] (C-Pt2), trans-[Pt(DACH)(OX)(L3)(OH)] (C-Pt3), and trans-[Pt(DACH)(OX)(L3)(BZ)] (C-Pt4) (DACH: R,R-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand L3. Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of L3 in the TSPO binding site were studied using molecular docking. The results showed that complex C-Pt1 is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.

Analysis of novel missense ATR mutations reveals new splicing defects underlying Seckel syndrome.

Ataxia Telangiectasia and Rad3 related (ATR) is one of the main regulators of the DNA damage response. It coordinates cell cycle checkpoint activation, replication fork stability, restart and origin firing to maintain genome integrity. Mutations of the ATR gene have been reported in Seckel patients, who suffer from a rare genetic disease characterized by severe microcephaly and growth retardation. Here, we report the case of a Seckel patient with compound heterozygous mutations in ATR. One allele has an intronic mutation affecting splicing of neighboring exons, the other an exonic missense mutation, producing the variant p.Lys1665Asn, of unknown pathogenicity. We have modeled this novel missense mutation, as well as a previously described missense mutation p.Met1159Ile, and assessed their effect on ATR function. Interestingly, our data indicate that both missense mutations have no direct effect on protein function, but rather result in defective ATR splicing. These results emphasize the importance of splicing mutations in Seckel Syndrome.

A function for ataxia telangiectasia and Rad3-related (ATR) kinase in cytokinetic abscission.

Abscission, the final stage of cytokinesis, occurs when the cytoplasmic canal connecting two emerging daughter cells is severed either side of a large proteinaceous structure, the midbody. Here, we expand the functions of ATR to include a cell-cycle-specific role in abscission, which is required for genome stability. All previously characterized roles for ATR depend upon its recruitment to replication protein A (RPA)-coated single-stranded DNA (ssDNA). However, we establish that in each cell cycle ATR, as well as ATRIP, localize to the midbody specifically during late cytokinesis and independently of RPA or detectable ssDNA. Rather, midbody localization and ATR-dependent regulation of abscission requires the known abscission regulator-charged multivesicular body protein 4C (CHMP4C). Intriguingly, this regulation is also dependent upon the CDC7 kinase and the known ATR activator ETAA1. We propose that in addition to its known RPA-ssDNA-dependent functions, ATR has further functions in preventing premature abscission.

The nuclear kinesin KIF18B promotes 53BP1-mediated DNA double-strand break repair.

53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.

A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining.

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an 'effector' protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.