Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that ...induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.
Of the many types of DNA damage, DNA double-strand breaks (DSBs) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system ...in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Here in a human cell line, we map genome-wide and at high resolution the DSBs induced by a restriction enzyme, and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and on the distance from the DSB. Our study couples for the first time, to the best of our knowledge, high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites.
The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. ...Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.
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•Purified MRE11-RAD50-NBS1 and RNAPII suffice to reconstitute dilncRNA synthesis•dilncRNA synthesis by RNAPII depends on the ability of MRN to melt DNA ends•RPA enhances MRN-dependent dilncRNA synthesis from DNA ends•Unpaired DNA ends allow MRN-independent dilncRNA synthesis
Sharma et al. show that in an in vitro reconstituted system, the DNA damage-sensing complex MRE11-RAD50-NBS1 (MRN) and RNA polymerase II are sufficient to synthesize RNA transcripts from broken DNA ends. MRN supports transcription by melting DNA ends and allowing RNA polymerase II to initiate RNA synthesis.
Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors ...with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.
The synthesis and characterization of four novel donor-acceptor-donor π-extended oligomers, incorporating naphtha(1-b)thiophene-4-carboxylate or benzo(b)thieno(3,2-g) benzothiophene-4-carboxylate ...2-octyldodecyl esters as end-capping moieties, and two different conjugated core fragments, is reported. The end-capping moieties are obtained via a cascade sequence of sustainable organic reactions, and then coupled to benzo(c)(1,2,5)thiadiazole and its difluoro derivative as the electron-poor π-conjugated cores. The optoelectronic properties of the oligomers are reported. The novel compounds revealed good film forming properties, and when tested in bulk-heterojunction organic photovoltaic cell devices in combination with PC
BM, revealed good fill factors, but low efficiencies, due to their poor absorption profiles.
The synthesis of several difunctional monomers, derived from the introduction of two 4-styrene-substituted moieties into a covalent skeleton, is described. The more rigid ones, built around malonate ...moieties and more preorganized to give 3,3′-cyclophane repeating units, failed completely to give soluble, ordered cyclopolymers. The introduction of a certain degree of flexibility in the tethering moiety afforded, by using free-radical initiation, structurally stable cyclopolymers with a high degree of cyclization and good degrees of polymerization. Theoretical calculations detail and strongly support the experimental results. Further chemical elaboration of the obtained cyclopolymeric backbones, by means of the removal of a ketal group, is possible in mild conditions to give systems which can be easily cross-linked thermally, with the loss of H2O, at temperatures higher than 100 °C.
Thiol-ene/−yne click reactions can be a versatile toolbox for the design and modification of polymers, enhancing functionality and providing opportunities for the development of innovative materials ...and applications. Here, we report a simple strategy to synthesize alkoxysilyl telechelic poly(propylene oxide)s (PPO)s: a) through a post-polymerization functionalization via Williamson reactions, commercial PPO is modified at the chain-end secondary alcohols with allyl or propargyl units, achieving >99% conversion; b) subsequent click reactions of (3-mercaptopropyl)trimethoxysilane (MPTMS), triggered by thermal and photochemical radical initiators, result in the incorporation of alkoxysilane groups on the polyether chain. Both thermal and photochemical thiol-ene/−yne click pathways are robust methodologies for preparing alkoxysilyl-modified PPOs in excellent yields (> 99%). Optimal conditions were identified as solvent-free, at 65 °C with 2,2′-azobis(2-methylpropionitrile) (AIBN) as the radical source. Detailed NMR analyses confirm the quantitative transformation of vinyl/propargyl functionalities in the resulting alkoxysilyl telechelic PPOs. Preliminary studies on the stability of alkoxysilyl end-groups are reported using 29Si NMR spectroscopy.
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•(Multi)functional alkoxysilyl-modified polyethers were prepared and characterized.•Solvent-free thiol-ene/−yne click processes•Quantitative evaluation of the degree of end-functionalization by NMR spectroscopy•The resulting silyl-functionalized polymers showed good stability over time.
Summary
The DNA damage response (DDR) arrests cell cycle progression until DNA lesions, like DNA double‐strand breaks (DSBs), are repaired. The presence of DSBs in cells is usually detected by ...indirect techniques that rely on the accumulation of proteins at DSBs, as part of the DDR. Such detection may be biased, as some factors and their modifications may not reflect physical DNA damage. The dependency on DDR markers of DSB detection tools has left questions unanswered. In particular, it is known that senescent cells display persistent DDR foci, that we and others have proposed to be persistent DSBs, resistant to endogenous DNA repair activities. Others have proposed that these peculiar DDR foci might not be sites of damaged DNA per se but instead stable chromatin modifications, termed DNA‐SCARS. Here, we developed a method, named ‘DNA damage in situ ligation followed by proximity ligation assay’ (DI‐PLA) for the detection and imaging of DSBs in cells. DI‐PLA is based on the capture of free DNA ends in fixed cells in situ, by ligation to biotinylated double‐stranded DNA oligonucleotides, which are next recognized by antibiotin anti‐bodies. Detection is enhanced by PLA with a partner DDR marker at the DSB. We validated DI‐PLA by demonstrating its ability to detect DSBs induced by various genotoxic insults in cultured cells and tissues. Most importantly, by DI‐PLA, we demonstrated that both senescent cells in culture and tissues from aged mammals retain true unrepaired DSBs associated with DDR markers.
•We review the roles of the evolutionarily conserved checkpoint kinases ATM/Tel1 and ATR/Mec1 in the response to DNA double-strand breaks (DSBs).•We give a summary of the current literature on DSB ...processing.•We discuss how ATM/Tel1 and ATR/Mec1 are activated by DNA double-strand breaks.•We discuss the interplays between ATM/Tel1 and ATR/Mec1 signaling activities at DNA ends.
DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.
Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double‐strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA‐coated single‐stranded DNA (ssDNA), which arises ...from 5′–3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1‐ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53‐dependent and Rad53‐independent mechanisms. The mec1‐ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.
Synopsis
Persistent Tel1 activation upon defective Mec1‐dependent resection in yeast interferes with checkpoint adaptation, exemplifying a role for the ATM/ATR switch previously observed in human cells.
A Mec1‐ad mutant variant that impaired resection of DNA ends was identified by searching for mec1 mutants failing to turn off the checkpoint after generation of a single irreparable DNA double‐strand break (DSB).
Mec1‐ad affects DSB resection by increasing the efficiency of γH2A generation, which in turn causes enhanced amount/persistence of the resection inhibitor Rad9 at DNA ends.
Defective DSB resection caused by either Rad9 excess or dysfunctions of the resection machinery leads to persistent Tel1‐dependent checkpoint signaling by prolonging MRX occupancy at DSBs.
Switch from Tel1‐ to Mec1‐dependent signaling activity promoted by DSB resection ensures proper termination of the checkpoint response.
Persistent Tel1 activation upon defective Mec1‐dependent resection in yeast interferes with checkpoint adaptation, exemplifying a role for the ATM/ATR switch previously observed in human cells.
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