<p>(A) Clonogenic survival after the indicated treatments of U2OS cells with ICRF-193 or Ol... more <p>(A) Clonogenic survival after the indicated treatments of U2OS cells with ICRF-193 or Olaparib. (B) NHEJ and HR repair assays. Cell lines with stably integrated constructs specific for either NHEJ or HR were transfected with a plasmid that expresses the I-SceI endonuclease and with the indicated siRNAs. Cut sites and the EGFP gene have been transfected with a plasmid expressing the enzyme. Efficiency of reconstitution of the EGFP gene (measured by EGFP expression) is indicated. In both (A) and (B) error bars are the standard error of the mean (SEM) from three repetitions.</p
The design, synthesis, characterization and biological activity of a series of platinum(IV) pro-d... more The design, synthesis, characterization and biological activity of a series of platinum(IV) pro-drugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that bind 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 non-cancerous 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.
53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify t... more 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.
Abscission, the final stage of cytokinesis, occurs when the cytoplasmic canal con- necting two em... more Abscission, the final stage of cytokinesis, occurs when the cytoplasmic canal con- necting two emerging daughter cells is severed either side of a large proteina- ceous 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 pattern of transcription initiation for the human c-Ha-rasl gene has been investigated using ... more The pattern of transcription initiation for the human c-Ha-rasl gene has been investigated using several experimental methods. These analyses demonstrated multiple clusters of transcription initiation sites distributed over an approximately 200 bp non-coding, upstream exon (termed exon -1), which is separated from the ATG codon by an 1040 bp intron. Mutational analysis of the promoter region identified a short positive regulatory element, located between positions -243 to -196, relative to the donor splice site of exon -1. This element contains known regulatory sequence motifs. Furthermore, a putative negative regulatory element, with an unusual DNA sequence, was identified between positions -103 to -34, relative to the same donor splice site. It has also been demonstrated that the human c-Ha-rasl promoter region can function bidirectionally. The sequence directing the "reverse-orientation" promoter activity was located to between positions, -392 to -196, relative to the e...
<p>Upon damage (step I) ATM is activated and recruited to chromatin containing a DSB (step ... more <p>Upon damage (step I) ATM is activated and recruited to chromatin containing a DSB (step II). One of the substrates of ATM is the RSF1 protein. Once activated by ATM-dependent phosphorylation of RSF1 (step III), we propose that the RSF chromatin-remodelling complex facilitates DSB repair utilising its translocase activity, via an ill-understood mechanism likely to involve nucleosome sliding (step III) and even nucleosome clearance (step IV). As part of this process, CENPS/MHF1 and CENPX/MHF2, two histone-fold proteins, are recruited to the vicinity of the DSB in an RSF-dependent fashion (step V). As the CENPS/MHF1–CENPX/MHF2 tetramer has fewer contacts with DNA than a nucleosome or the H3–H4 tetramer <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio.1001856-Yang1" target="_blank">[25]</a>, it could perform a “<i>placeholder</i>” function, preventing DNA that has been unwound from nucleosomes on either side of the DSB from forming a random coil and also facilitating the subsequent access of DSB repair machineries. Downstream of CENPS/MHF1 and CENPX/MHF2 recruitment, the FANCD2 and FANCI proteins are recruited and mono-ubiquitinated by a mechanism dependent upon prior recruitment of CENPS/MHF1 and CENPX/MHF2 (step 6). It is not know how many steps exist between CENPS/MHF1–CENPX/MHF2 and subsequent FANCD2–FANCI recruitment. Mono-ubiquitinated FANCD2 and FANCI presumably functions as docking sites that regulate recruitment of subsequent DSB repair proteins, such as CtIP, which has functions in both NHEJ and HR.</p
<p>(A) Co-immunoprecipitation (note that benzonase was included during cell lysis to solubi... more <p>(A) Co-immunoprecipitation (note that benzonase was included during cell lysis to solubilise chromatin-bound proteins; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#s4" target="_blank">Materials and Methods</a>) of the indicated proteins with ATM. U2OS cells were either mock treated or treated with 10 Gy IR and harvested 1 h after irradiation. Where indicated the ATM inhibitor KU55933 was added directly to the media an hour before irradiation. (B) RSF1 immunoprecipitation. Cells were mock treated or treated with 5 Gy of IR and 5 Gy of IR plus ATM inhibitor. Elutions were blotted with RSF1 monoclonal antibody (Millipore) and general SQ/TQ antibody (Cell Signalling). (C) Typical efficiency of RSF1 and SNF2H depletion (siRNA) in U2OS cells. (D) Clonogenic survival after the indicated treatments of U2OS cells with IR or MMS. (E) Recombinant GST fusion proteins expressing fragments of ATM proteins were purified by <i>E. coli</i>, and approximately 5 mg of each GST fusion protein was used to incubate with cell lysates from HEK293 cells that had been previously irradiated with 5 Gy of IR and harvested 1 h post-IR. Elutions were blotted for RSF1 together with NBS1 as a control <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio.1001856-Karlseder1" target="_blank">[37]</a>. Schematic adapted from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio.1001856-Perry1" target="_blank">[43]</a> shows heat repeats (black squares) conserved in the PIK family and heat repeats specific for ATM protein (blue squares); the domains are indicated, as are the regions where the GST fragments are mapping.</p
<p>(A) Schematic representation of the Rad9 N-terminus showing the 9 consensus sites for ph... more <p>(A) Schematic representation of the Rad9 N-terminus showing the 9 consensus sites for phosphorylation by Cdc28 and those mutated to alanine in Rad9<sup>CDK1-9A</sup> mutant protein. (B) <i>In vitro</i> phosphorylation of Rad9 CAD<sup>WT</sup>, but not CAD<sup>CDK1-9A</sup>, requires Cdc28/Clb2. <i>In vitro</i> kinase assays were performed on the indicated substrates with four specific purified Cdc28 complexes. (C) DNA damage-dependent Chk1 phosphorylation is defective in <i>rad9<sup>CDK1-9A</sup>, rad9<sup>CADΔ</sup></i> and <i>rad9 Δ</i> cells. Asynchronously growing cells were treated with bleocin for the indicated times and Chk1 phosphorylation analysed by western blotting. (D&E) Chk1 activation is mostly dependent on the 9 CAD CDK consensus sites in G2/M cells. Cells were grown and arrested in the cell cycle as indicated with either α-factor (G1 cells in D) or nocodazole (G2/M cells in E), treated with bleocin for the indicated times and analysed by western blotting of Chk1-3HA (D & E). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s002" target="_blank">Figure S2</a> for related experiments.</p
<p>(A) Rad9/Chk1 interaction measured <i>in vivo</i> using a yeast two-hybrid (... more <p>(A) Rad9/Chk1 interaction measured <i>in vivo</i> using a yeast two-hybrid (Y2H) assay is dependent on the CDK1-9 sites. Y2H interaction of specific bait and prey plasmids shown on the left is indicated by the white colour of the otherwise red cells, their resistance to Aureobasidin A and their blue colour on media containing the X-α-gal substrate, as for the p53/T antigen interaction control. Six independent clones are presented for each vector combination. (B) The Y2H interaction between Rad9 and Chk1 is dependent on CDK activity in G2/M cells. The indicated bait and prey plasmids were introduced into <i>cdc28-as1</i> cells mock treated or treated with 1-NMPP1 1 h after synchronization of cells with either nocodazole or alpha factor and prior to induction of expression of each bait protein. The recently reported CDK-dependent Rad9/Dpb11 interaction was used as a positive control. (C) The Rad9/Chk1 interaction measured using co-immunoprecipitation (co-IP) occurs both in the absence and presence of DNA damage. Chk1 (anti-FLAG) and Dpb11 (anti-MYC) immunoprecipitations (IPs) were performed as indicated on extracts prepared from nocodazole-arrested cells, expressing both Chk1-3FLAG and Dpb11-13MYC, and either mock treated or treated with 20 µg/ml of bleocin for 45 min. Mock (IgG) or Dpb11 (MYC) IPs were performed as controls. Rad9, Chk1-3FLAG and Dpb11-13MYC specific bands were detected in western blots. Lower exposures, to facilitate their visualisation, of the western blots of the starting extracts are shown to the left. (D) Chk1 interaction with Rad9 is dependent on the CDK1-9 sites. As in panel C, except <i>rad9<sup>CDK1-9A</sup></i> cells were used. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s005" target="_blank">Figure S5</a> for related experiments.</p
<p><i>rad9<sup>CDK1-9A</sup></i> mutant cells arrested in G2/M by n... more <p><i>rad9<sup>CDK1-9A</sup></i> mutant cells arrested in G2/M by nocodazole treatment are neither defective for Rad9 (A) nor for Rad53 (B) phosphorylations induced by bleocin treatment started at time 0. (C) The CDK1-9 sites of Rad9 function specifically in the Chk1 branch and not the Rad53 branch of the G2/M checkpoint. The indicated strains were examined for epistatic relationships using the G2/M checkpoint assay, in which cells synchronized in G2/M using nocodazole are released after irradiation into medium without nocodazole, but containing α-factor, preventing cells that have successfully completed mitosis from cycling further by arresting them in G1. This assay measures the delay in completing mitosis under DNA damaging conditions by comparing the behavior of cells that have been irradiated with IR (+IR) or not (−IR). All strains contained the <i>sml1Δ</i> mutation necessary for the viability of <i>rad53Δ</i> cells. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s003" target="_blank">Figure S3</a>.</p
<p>(A) G2/M checkpoint analysis in U2OS cells. Inhibition of ATM or specific siRNA depletio... more <p>(A) G2/M checkpoint analysis in U2OS cells. Inhibition of ATM or specific siRNA depletions are as indicated. Cells positive for the mitotic marker were followed for 48 h after irradiation. (B) Western blot analysis of γ-H2AX phosphorylation after IR (4 Gy) over 24 h in U2OS cells treated as indicated. The extent of knockdown is indicated in the RSF1 and SNF2H blots. (C) Immunofluorescence showing γ-H2AX IRIF formation and persistence in U2OS cells over 48 h postradiation (4 Gy) and treated with the indicated siRNAs. (D) Neutral comet assay showing the repair of fragmented DNA induced by 2 Gy of IR in U2OS cells treated either with an ATM inhibitor (KU55933) or with the indicated siRNAs. (E) Quantification of tail moments represented in the neutral comet assay shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio-1001856-g003" target="_blank">Figure 3D</a>. Error bars indicate standard error of the mean (SEM) from three independent experiments.</p
ABSTRACTProteins with RNA-binding activity are increasingly being implicated in DNA damage respon... more ABSTRACTProteins 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 are naturally deficient for DNA:RNA-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 ...
<p>Cdc28 activity was regulated using the 1-NMPP1 inhibitor in G2/M arrested <i>cdc28... more <p>Cdc28 activity was regulated using the 1-NMPP1 inhibitor in G2/M arrested <i>cdc28-as1</i> cells treated with bleocin or 4-NQO to examine the activation of Chk1 signaling. Rad9 and Rad53 were followed as markers of checkpoint activation, while Orc6 phosphorylation serves as a marker for Cdc28 inactivation. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s004" target="_blank">Figure S4</a>.</p
<p>(A) Clonogenic survival after the indicated treatments of U2OS cells with ICRF-193 or Ol... more <p>(A) Clonogenic survival after the indicated treatments of U2OS cells with ICRF-193 or Olaparib. (B) NHEJ and HR repair assays. Cell lines with stably integrated constructs specific for either NHEJ or HR were transfected with a plasmid that expresses the I-SceI endonuclease and with the indicated siRNAs. Cut sites and the EGFP gene have been transfected with a plasmid expressing the enzyme. Efficiency of reconstitution of the EGFP gene (measured by EGFP expression) is indicated. In both (A) and (B) error bars are the standard error of the mean (SEM) from three repetitions.</p
The design, synthesis, characterization and biological activity of a series of platinum(IV) pro-d... more The design, synthesis, characterization and biological activity of a series of platinum(IV) pro-drugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that bind 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 non-cancerous 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.
53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify t... more 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.
Abscission, the final stage of cytokinesis, occurs when the cytoplasmic canal con- necting two em... more Abscission, the final stage of cytokinesis, occurs when the cytoplasmic canal con- necting two emerging daughter cells is severed either side of a large proteina- ceous 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 pattern of transcription initiation for the human c-Ha-rasl gene has been investigated using ... more The pattern of transcription initiation for the human c-Ha-rasl gene has been investigated using several experimental methods. These analyses demonstrated multiple clusters of transcription initiation sites distributed over an approximately 200 bp non-coding, upstream exon (termed exon -1), which is separated from the ATG codon by an 1040 bp intron. Mutational analysis of the promoter region identified a short positive regulatory element, located between positions -243 to -196, relative to the donor splice site of exon -1. This element contains known regulatory sequence motifs. Furthermore, a putative negative regulatory element, with an unusual DNA sequence, was identified between positions -103 to -34, relative to the same donor splice site. It has also been demonstrated that the human c-Ha-rasl promoter region can function bidirectionally. The sequence directing the "reverse-orientation" promoter activity was located to between positions, -392 to -196, relative to the e...
<p>Upon damage (step I) ATM is activated and recruited to chromatin containing a DSB (step ... more <p>Upon damage (step I) ATM is activated and recruited to chromatin containing a DSB (step II). One of the substrates of ATM is the RSF1 protein. Once activated by ATM-dependent phosphorylation of RSF1 (step III), we propose that the RSF chromatin-remodelling complex facilitates DSB repair utilising its translocase activity, via an ill-understood mechanism likely to involve nucleosome sliding (step III) and even nucleosome clearance (step IV). As part of this process, CENPS/MHF1 and CENPX/MHF2, two histone-fold proteins, are recruited to the vicinity of the DSB in an RSF-dependent fashion (step V). As the CENPS/MHF1–CENPX/MHF2 tetramer has fewer contacts with DNA than a nucleosome or the H3–H4 tetramer <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio.1001856-Yang1" target="_blank">[25]</a>, it could perform a “<i>placeholder</i>” function, preventing DNA that has been unwound from nucleosomes on either side of the DSB from forming a random coil and also facilitating the subsequent access of DSB repair machineries. Downstream of CENPS/MHF1 and CENPX/MHF2 recruitment, the FANCD2 and FANCI proteins are recruited and mono-ubiquitinated by a mechanism dependent upon prior recruitment of CENPS/MHF1 and CENPX/MHF2 (step 6). It is not know how many steps exist between CENPS/MHF1–CENPX/MHF2 and subsequent FANCD2–FANCI recruitment. Mono-ubiquitinated FANCD2 and FANCI presumably functions as docking sites that regulate recruitment of subsequent DSB repair proteins, such as CtIP, which has functions in both NHEJ and HR.</p
<p>(A) Co-immunoprecipitation (note that benzonase was included during cell lysis to solubi... more <p>(A) Co-immunoprecipitation (note that benzonase was included during cell lysis to solubilise chromatin-bound proteins; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#s4" target="_blank">Materials and Methods</a>) of the indicated proteins with ATM. U2OS cells were either mock treated or treated with 10 Gy IR and harvested 1 h after irradiation. Where indicated the ATM inhibitor KU55933 was added directly to the media an hour before irradiation. (B) RSF1 immunoprecipitation. Cells were mock treated or treated with 5 Gy of IR and 5 Gy of IR plus ATM inhibitor. Elutions were blotted with RSF1 monoclonal antibody (Millipore) and general SQ/TQ antibody (Cell Signalling). (C) Typical efficiency of RSF1 and SNF2H depletion (siRNA) in U2OS cells. (D) Clonogenic survival after the indicated treatments of U2OS cells with IR or MMS. (E) Recombinant GST fusion proteins expressing fragments of ATM proteins were purified by <i>E. coli</i>, and approximately 5 mg of each GST fusion protein was used to incubate with cell lysates from HEK293 cells that had been previously irradiated with 5 Gy of IR and harvested 1 h post-IR. Elutions were blotted for RSF1 together with NBS1 as a control <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio.1001856-Karlseder1" target="_blank">[37]</a>. Schematic adapted from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio.1001856-Perry1" target="_blank">[43]</a> shows heat repeats (black squares) conserved in the PIK family and heat repeats specific for ATM protein (blue squares); the domains are indicated, as are the regions where the GST fragments are mapping.</p
<p>(A) Schematic representation of the Rad9 N-terminus showing the 9 consensus sites for ph... more <p>(A) Schematic representation of the Rad9 N-terminus showing the 9 consensus sites for phosphorylation by Cdc28 and those mutated to alanine in Rad9<sup>CDK1-9A</sup> mutant protein. (B) <i>In vitro</i> phosphorylation of Rad9 CAD<sup>WT</sup>, but not CAD<sup>CDK1-9A</sup>, requires Cdc28/Clb2. <i>In vitro</i> kinase assays were performed on the indicated substrates with four specific purified Cdc28 complexes. (C) DNA damage-dependent Chk1 phosphorylation is defective in <i>rad9<sup>CDK1-9A</sup>, rad9<sup>CADΔ</sup></i> and <i>rad9 Δ</i> cells. Asynchronously growing cells were treated with bleocin for the indicated times and Chk1 phosphorylation analysed by western blotting. (D&E) Chk1 activation is mostly dependent on the 9 CAD CDK consensus sites in G2/M cells. Cells were grown and arrested in the cell cycle as indicated with either α-factor (G1 cells in D) or nocodazole (G2/M cells in E), treated with bleocin for the indicated times and analysed by western blotting of Chk1-3HA (D & E). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s002" target="_blank">Figure S2</a> for related experiments.</p
<p>(A) Rad9/Chk1 interaction measured <i>in vivo</i> using a yeast two-hybrid (... more <p>(A) Rad9/Chk1 interaction measured <i>in vivo</i> using a yeast two-hybrid (Y2H) assay is dependent on the CDK1-9 sites. Y2H interaction of specific bait and prey plasmids shown on the left is indicated by the white colour of the otherwise red cells, their resistance to Aureobasidin A and their blue colour on media containing the X-α-gal substrate, as for the p53/T antigen interaction control. Six independent clones are presented for each vector combination. (B) The Y2H interaction between Rad9 and Chk1 is dependent on CDK activity in G2/M cells. The indicated bait and prey plasmids were introduced into <i>cdc28-as1</i> cells mock treated or treated with 1-NMPP1 1 h after synchronization of cells with either nocodazole or alpha factor and prior to induction of expression of each bait protein. The recently reported CDK-dependent Rad9/Dpb11 interaction was used as a positive control. (C) The Rad9/Chk1 interaction measured using co-immunoprecipitation (co-IP) occurs both in the absence and presence of DNA damage. Chk1 (anti-FLAG) and Dpb11 (anti-MYC) immunoprecipitations (IPs) were performed as indicated on extracts prepared from nocodazole-arrested cells, expressing both Chk1-3FLAG and Dpb11-13MYC, and either mock treated or treated with 20 µg/ml of bleocin for 45 min. Mock (IgG) or Dpb11 (MYC) IPs were performed as controls. Rad9, Chk1-3FLAG and Dpb11-13MYC specific bands were detected in western blots. Lower exposures, to facilitate their visualisation, of the western blots of the starting extracts are shown to the left. (D) Chk1 interaction with Rad9 is dependent on the CDK1-9 sites. As in panel C, except <i>rad9<sup>CDK1-9A</sup></i> cells were used. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s005" target="_blank">Figure S5</a> for related experiments.</p
<p><i>rad9<sup>CDK1-9A</sup></i> mutant cells arrested in G2/M by n... more <p><i>rad9<sup>CDK1-9A</sup></i> mutant cells arrested in G2/M by nocodazole treatment are neither defective for Rad9 (A) nor for Rad53 (B) phosphorylations induced by bleocin treatment started at time 0. (C) The CDK1-9 sites of Rad9 function specifically in the Chk1 branch and not the Rad53 branch of the G2/M checkpoint. The indicated strains were examined for epistatic relationships using the G2/M checkpoint assay, in which cells synchronized in G2/M using nocodazole are released after irradiation into medium without nocodazole, but containing α-factor, preventing cells that have successfully completed mitosis from cycling further by arresting them in G1. This assay measures the delay in completing mitosis under DNA damaging conditions by comparing the behavior of cells that have been irradiated with IR (+IR) or not (−IR). All strains contained the <i>sml1Δ</i> mutation necessary for the viability of <i>rad53Δ</i> cells. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s003" target="_blank">Figure S3</a>.</p
<p>(A) G2/M checkpoint analysis in U2OS cells. Inhibition of ATM or specific siRNA depletio... more <p>(A) G2/M checkpoint analysis in U2OS cells. Inhibition of ATM or specific siRNA depletions are as indicated. Cells positive for the mitotic marker were followed for 48 h after irradiation. (B) Western blot analysis of γ-H2AX phosphorylation after IR (4 Gy) over 24 h in U2OS cells treated as indicated. The extent of knockdown is indicated in the RSF1 and SNF2H blots. (C) Immunofluorescence showing γ-H2AX IRIF formation and persistence in U2OS cells over 48 h postradiation (4 Gy) and treated with the indicated siRNAs. (D) Neutral comet assay showing the repair of fragmented DNA induced by 2 Gy of IR in U2OS cells treated either with an ATM inhibitor (KU55933) or with the indicated siRNAs. (E) Quantification of tail moments represented in the neutral comet assay shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001856#pbio-1001856-g003" target="_blank">Figure 3D</a>. Error bars indicate standard error of the mean (SEM) from three independent experiments.</p
ABSTRACTProteins with RNA-binding activity are increasingly being implicated in DNA damage respon... more ABSTRACTProteins 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 are naturally deficient for DNA:RNA-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 ...
<p>Cdc28 activity was regulated using the 1-NMPP1 inhibitor in G2/M arrested <i>cdc28... more <p>Cdc28 activity was regulated using the 1-NMPP1 inhibitor in G2/M arrested <i>cdc28-as1</i> cells treated with bleocin or 4-NQO to examine the activation of Chk1 signaling. Rad9 and Rad53 were followed as markers of checkpoint activation, while Orc6 phosphorylation serves as a marker for Cdc28 inactivation. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003310#pgen.1003310.s004" target="_blank">Figure S4</a>.</p
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Papers by Noel Lowndes