The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection.

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5'-3' degradation of DSB ends that yields the 3' single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11-Rad50-Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 together with the endonuclease Dna2 (refs 1-6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.

Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling.

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.

Climate footprint of industry-sponsored clinical research: an analysis of a phase-1 randomised clinical study and discussion of opportunities to reduce its impact.

OBJECTIVE: This study aims to calculate the global warming potential, in carbon dioxide (CO2) equivalent emissions, from all in-scope activities involved in a phase-1 clinical study. DESIGN: Retrospective analysis. DATA SOURCE: Internal data held by Janssen Pharmaceuticals. STUDIES INCLUDED: Janssen-sponsored TMC114FD1HTX1002 study conducted between 2019 and 2021. MAIN OUTCOME: Measure CO2 equivalents (CO2e) for in-scope clinical trial activities calculated according to intergovernmental panel on climate change 2021 impact assessment methodology. RESULTS: The CO2e emissions generated by the trial were 17.65 tonnes. This is equivalent to the emissions generated by driving an average petrol-fueled family car 71 004 km or roughly 1.8 times around the circumference of the Earth. Commuting to the clinical site by the study participants generated the most emissions (5419 kg, 31% of overall emissions), followed by trial site utilities (2725 kg, 16% of overall emissions) and site staff travel (2560 kg, 15% of overall emissions). In total, the movement of people (participant travel, site staff travel and trial site staff travel) accounted for 8914 kg or 51% of overall trial emissions. CONCLUSIONS: Decentralised trial models which seek to bring clinical trial operations closer to the participant offer opportunities to reduce participant travel. The electrification of sponsor vehicle fleets and society's transition towards electric vehicles may result in further reductions. TRIAL REGISTRATION NUMBER: NCT04208061.

In Silico Assessment of Biomolecule Reactivity with Leachables.

Leachables in pharmaceutical products may react with biomolecule active pharmaceutical ingredients (APIs), for example, monoclonal antibodies (mAb), peptides, and ribonucleic acids (RNA), potentially compromising product safety and efficacy or impacting quality attributes. This investigation explored a series of in silico models to screen extractables and leachables to assess their possible reactivity with biomolecules. These in silico models were applied to collections of known leachables to identify functional and structural chemical classes likely to be flagged by these in silico approaches. Flagged leachable functional classes included antimicrobials, colorants, and film-forming agents, whereas specific chemical classes included epoxides, acrylates, and quinones. In addition, a dataset of 22 leachables with experimental data indicating their interaction with insulin glargine was used to evaluate whether one or more in silico methods are fit-for-purpose as a preliminary screen for assessing this biomolecule reactivity. Analysis of the data showed that the sensitivity of an in silico screen using multiple methodologies was 80%-90% and the specificity was 58%-92%. A workflow supporting the use of in silico methods in this field is proposed based on both the results from this assessment and best practices in the field of computational modeling and quality risk management.

Carbon footprint of industry-sponsored late-stage clinical trials.

OBJECTIVES: To quantify the carbon footprint from a sample of pharma industry sponsored phase III trials. To develop an approach that can readily be applied to future trials by AstraZeneca and other trial sponsors. DESIGN: Life cycle assessment including all the sources of carbon emissions associated with a completed, an ongoing and a planned clinical trial. The methodology followed the guidance on appraising the sustainability of Care Pathways, developed by the UK National Health Service in collaboration with parties across the healthcare system. SETTING: Three multicentre late phase trials. One completed heart failure trial, one ongoing oncology trial and one asthma trial with the addition of devices to be representative of current practice. PARTICIPANTS: The three trials had a total number of 7412 participants. MAIN OUTCOME MEASURES: Total carbon emissions from each trial, the drivers of those emissions and the emissions per patient. RESULTS: The total carbon footprint for the cardiovascular trial was calculated as 2498 tonnes carbon dioxide equivalents (CO2e), the first 3 years of the oncology trial resulted in 1632 tonnes CO2e and the respiratory trial 1437 tonnes CO2e. CONCLUSIONS: We have shown that it is feasible to perform a retrospective life cycle assessment to appraise the carbon footprint of large clinical trials and confirmed that phase III trials result in significant emissions. Having identified all the drivers of emissions and their magnitude, we are well placed to develop a plan for achieving net-zero carbon clinical trials. Now it is possible to expand the use of life cycle assessment to planned studies so that scientific aims can be achieved with a minimum of carbon emissions. We encourage other trialists to apply the same methodology as a necessary first step in reducing the carbon footprint of clinical trials.

Chromatin assembly and signalling the end of DNA repair requires acetylation of histone H3 on lysine 56.

The packaging of DNA into chromatin results in a barrier to all DNA transactions. To facilitate transcription, replication and repair histone proteins are frequently post-translational modified. Such covalent additions to histone residues can modulate chromatin folding and/or provide specificity to docking surfaces for non-histone chromatin proteins. In the budding yeast, one such modification, transient acetylation of histone H3 on residue lysine 56 (H3K56ac); occurs on newly synthesized H3 molecules and facilitates their deposition onto newly replicated DNA during S phase. H3K56ac also has a role in chromatin reassembly following DNA damage in S phase. Importantly, the completion of H3K56ac-dependent chromatin reassembly appears to be required for resumption of cell proliferation after DNA repair. Emerging evidence, although not without conflict, suggests that H3K56ac is not only present in human cells, but is similarly regulated and required for chromatin reassembly.

A UV-induced genetic network links the RSC complex to nucleotide excision repair and shows dose-dependent rewiring.

Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

Docking onto chromatin via the Saccharomyces cerevisiae Rad9 Tudor domain.

An integrated cellular response to DNA damage is essential for the maintenance of genome integrity. Recently, post-translational modifications to histone proteins have been implicated in DNA damage responses involving the Rad9 family of checkpoint proteins. In budding yeast, methylation of histone H3 on lysine 79 (H3-K79me) has been shown to be required for efficient checkpoint signalling and Rad9 localization on chromatin. Here, we have used a rad9 Tudor mutant allele and cells mutated for Dot1, the H3-K79 methylase, to analyse the epistatic relationship between RAD9 and DOT1 genes regarding the DNA damage resistance and checkpoint activation pathways. Our results show that RAD9 is epistatic to DOT1 and suggest that it acts downstream of the Dot1 methylase in the damage resistance and checkpoint response. We have also found that the Tudor domain of Rad9 is necessary for in vitro binding to H3-K79me as well as Rad9 focal accumulation in response to DNA damage in vivo. In summary, our study demonstrates that the interaction between Rad9, via its Tudor domain, and methylated H3-K79 is required at two different steps of the DNA damage response, an early step corresponding to checkpoint activation, and a late step corresponding to DNA repair. The study further shows that the function of this interaction is cell cycle-regulated; the role in checkpoint activation is restricted to the G(1) phase and its role in DNA repair is restricted to G(2).

Chromatin modulation and the DNA damage response.

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.