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Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability., (© 2024. The Author(s).)

Eukaryotic nuclei are structured into sub-compartments orchestrating various cellular functions. The nucleolus is the largest nuclear organelle: a biomolecular condensate with an architecture composed of immiscible fluids facilitating ribosome biogenesis. The nucleolus forms upon the transcription of the repetitive ribosomal RNA genes (rDNA) that cluster in this compartment. rDNA is intrinsically unstable and prone to rearrangements and copy number variation. Upon DNA damage, a specialized nucleolar-DNA Damage Response (n-DDR) is activated: nucleolar transcription is inhibited, the architecture is rearranged, and rDNA is relocated to the nucleolar periphery. Recent data have highlighted how the composition of nucleoli, its structure, chemical and physical properties, contribute to rDNA stability. In this mini-review we focus on recent data that start to reveal how nucleolar composition and the n-DDR work together to ensure rDNA integrity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The role of MDC1 in the DNA damage response has been extensively studied; however, its impact on other cellular processes is not well understood. Here, we describe the role of MDC1 in transcription as a regulator of RNA polymerase II (RNAPII). Depletion of MDC1 causes a genome-wide reduction in the abundance of actively engaged RNAPII elongation complexes throughout the gene body of protein-encoding genes under unperturbed conditions. Decreased engaged RNAPII subsequently alters the assembly of the spliceosome complex on chromatin, leading to changes in pre-mRNA splicing. Mechanistically, the S/TQ domain of MDC1 modulates RNAPII-mediated transcription. Upon genotoxic stress, MDC1 promotes the abundance of engaged RNAPII complexes at DNA breaks, thereby stimulating nascent transcription at the damaged sites. Of clinical relevance, cancer cells lacking MDC1 display hypersensitivity to RNAPII inhibitors. Overall, we unveil a role of MDC1 in RNAPII-mediated transcription with potential implications for cancer treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The relations between physical and chemical characteristics (e.g., color, firmness, volatile and non-volatile metabolites) of red ripe strawberry fruit and the natural spoilage caused by Botrytis cinerea were investigated. The spoilage rates differed between genotypes, and this was highly correlated over two successive years. Among seventeen genotypes, a more intense red coloration of the fruit skin was associated with a lower spoilage rate (r = -0.63). Additionally, weakly negative correlations were found between the levels of anthocyanins, ascorbic acid, malic acid and spoilage rates. No clear correlations were found between spoilage rates and soluble sugars, most volatiles, firmness and dry weight percentage. High levels of two volatile compounds, ethyl butanoate (r = 0.55) and 1-hexanol (r = 0.61), were correlated to high spoilage rates. These characteristics may assist strawberry breeders in selecting for genotypes with reduced susceptibility to B. cinerea., (Copyright © 2022. Published by Elsevier Ltd.)

Treacle/TCOF1 is an adaptor protein specifically associated with nucleolar chromatin. In the nucleolus it stimulates ribosome biogenesis, thereby promoting growth and proliferation. A second role of Treacle has emerged as a coordinator of the nucleolar responses to DNA damage, where it facilitates nucleolar DNA repair and cellular survival after genotoxic insults. The involvement of Treacle in multiple fundamental processes such as growth, proliferation, and genome stability, which are tightly linked to cancer, raises the question of Treacle's role in the development of this disease. On one hand, overexpression of Treacle could stimulate nucleolar transcription and ribosome biogenesis providing a growth advantage in cancer cells. On the other hand, the function of Treacle as a gatekeeper in response to nucleolar DNA damage could favor mutations that would impair its function. In this perspective, we analyze paired Treacle expression data from the Cancer Genome Atlas (TCGA) and correlate expression with patient survival in different cancer types. We also discuss other recently published observations of relevance to the role of Treacle in cancer. In light of these new observations, we propose possible roles of Treacle in carcinogenesis and discuss its potential as a therapeutic target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Oxe and Larsen.)

The importance of chromatin environment for DNA repair has gained increasing recognition in recent years. The nucleolus is the largest sub-compartment within the nucleus: it has distinct biophysical properties, selective protein retention, and houses the specialized ribosomal RNA genes (collectively referred to as rDNA) with a unique chromatin composition. These genes have high transcriptional activity and a repetitive nature, making them susceptible to DNA damage and resulting in the highest frequency of rearrangements across the genome. A distinct DNA damage response (DDR) secures the fidelity of this genomic region, the so-called nucleolar DDR (n-DDR). The composition of the n-DDR reflects the characteristics of nucleolar chromatin with the nucleolar protein Treacle (also referred to as TCOF1) as a central coordinator retaining several well-characterized DDR proteins in the nucleolus. In this review, we bring together data on the structure of Treacle, its known functions in ribosome biogenesis, and its involvement in multiple branches of the n-DDR to discuss their interconnection. Furthermore, we discuss how the functions of Treacle in ribosome biogenesis and in the n-DDR may contribute to Treacher Collins Syndrome, a disease caused by mutations in Treacle. Finally, we outline outstanding questions that need to be addressed for a more comprehensive understanding of Treacle, the n-DDR, and the coordination of ribosome biogenesis and DNA repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gál, Nieto, Boukoura, Rasmussen and Larsen.)

Blue light, measuring from 400 to 500 nm, is generally assumed to increase the content of antioxidants in plants independent of the species. Blue light stimulates the biosynthesis of phenolic compounds such as flavonoids and their subclass anthocyanins from the phenylpropanoid pathway. Flavonoids, anthocyanins, and phenolic acids are strong reactive oxygen species (ROS) scavengers and may lessen the symptoms of abiotic stresses such as chilling. We tested the hypothesis that a high percentage of blue light induces the accumulation of antioxidants and that this effect depends on the photosynthetic photon flux density (PPFD, 400-700 nm). The effect may be more pronounced at a lower PPFD. We investigated the changes in primary and secondary metabolites of basil in response to the percentage of blue light (9, 33, 65, and 100%) applied either as a 5-day End-Of-Production (EOP) treatment or continuous throughout the growth cycle in the green cv. Dolly. We also studied if the response to the percentage of blue light (9 or 90%) was dependent on the total PPFD (100 or 300 μmol m -2 s -1 PPFD) when applied as a 5-day EOP treatment in the green cv. Dolly and the purple cv. Rosie. For both green and purple basil, it was found that the percentage of blue light had little effect on the levels of antioxidants (rosmarinic acid, total ascorbic acid, total flavonoids, and total anthocyanins) at harvest and no interactive effect with PPFD was found. Antioxidants generally decreased during postharvest storage, wherein the decrease was more pronounced at 4 than at 12°C. Chilling injury, as judged from a decrease in F v /F m values and from the occurrence of black necrotic areas, was not affected by the percentage of blue light. Particularly, chilling tolerance in the purple cultivar was increased in plants grown under higher PPFD. This may be related to the increased levels of soluble sugar and starch in leaves from high PPFD treated plants., Competing Interests: CN was employed by Signify Research Laboratories Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Larsen, Li, Shrestha, Verdonk, Nicole, Marcelis and Woltering.)

Basil suffers from chilling injury (CI) when stored at temperatures below 10-12 °C which seems related to the imbalance between reactive oxygen species (ROS) and antioxidants. We hypothesized that increased light intensity applied shortly before harvest (EOP, End-Of-Production) increases nutritional value i.e. carbohydrates and antioxidants and could improve the chilling tolerance. Two basil cultivars were grown in a vertical farming set-up at a light intensity of 150 µmol m -2 s -1 . During the last 5 days of growth, EOP light treatments ranging from 50 to 600 µmol m -2 s -1 were applied. After harvest the leaves were stored at 4 or 12 °C in darkness. Higher EOP light intensity increased the antioxidant (total ascorbic acid, rosmarinic acid) and carbohydrate contents at harvest. During storage antioxidants decreased more rapidly at 4 than at 12 °C. However, increased EOP light intensity did not alleviate chilling symptoms suggesting a minor role of antioxidants studied against chilling stress., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Vertical farming is becoming increasingly popular for production of leafy vegetables and herbs, with basil ( Ocimum basilicum L.) as one of the most popular herbs. In basil most research has focused on increasing secondary metabolites with light spectra. However, knowledge about the effect of light intensity (photosynthetic photon flux density, PPFD) and spectra on growth and morphology is key for optimizing quality at harvest. The impact of PPFD and spectrum on plant growth and development is species dependent and currently few studies in basil are available. Understanding the response to End-Of-Production (EOP) light of growth and morphology is important for successful vertical farming. We performed a comprehensive series of experiments, where the effects of EOP PPFD, fraction of blue and their interaction on the growth and morphology were analyzed in two green and one purple basil cultivar. In addition, the impact of different EOP intensities and duration of far-red were investigated. We found that increasing the PPFD increased fresh mass, dry matter content and plant height in all three cultivars. The responses were linear or quadratic depending on the cultivar. A high fraction of blue (>90%) increased plant height and decreased the dry mass partitioning to the leaves. The only interaction found between the fraction of blue and overall PPFD was on plant height in the green cultivar whereas other growth parameters and morphology responded stronger to PPFD than to the fraction of blue light. Plant dry matter production was increased with the addition of far-red. Far-red EOP intensity treatments enhanced the fraction of dry mass partitioned to the leaves, whereas a prolonged far-red treatment enhanced partitioning to the stem. Both plant fresh mass and dry matter content were improved by applying high PPFD shortly before harvest. Light spectra were found to be of less importance than PPFD with respect to plant dry matter content. Light use efficiency (LUE) based on fresh mass decreased with increasing PPFD whereas LUE based on dry mass increased with increasing PPFD, when given as EOP treatments. The overall physiological mechanisms of the light intensity and spectral effects are discussed., Competing Interests: CS Nicole was employed by company Signify. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Larsen, Woltering, Nicole and Marcelis.)

Far-red (FR) light promotes fruit growth by increasing dry mass partitioning to fruits, but the mechanism behind this is unknown. We hypothesise that it is due to an increased fruit sink strength as FR radiation enhances sugar transportation and metabolism. Tomato plants were grown with or without 50-80 μmol m -2  s -1 of FR radiation added to a common background 150-170 μmol m -2  s -1 red + blue light-emitting diode lighting. Potential fruit growth, achieved by pruning each truss to one remaining fruit, was measured to quantify fruit sink strength. Model simulation was conducted to test whether the measured fruit sink strength quantitatively explained the FR effect on dry mass partitioning. Starch, sucrose, fructose and glucose content were measured. Expression levels of key genes involved in sugar transportation and metabolism were determined. FR radiation increased fruit sink strength by 38%, which, in model simulation, led to an increased dry mass partitioned to fruits that quantitatively agreed very well with measured partitioning. FR radiation increased fruit sugar concentration and upregulated the expression of genes associated with both sugar transportation and metabolism. This is the first study to demonstrate that FR radiation stimulates dry mass partitioning to fruits mainly by increasing fruit sink strength via simultaneous upregulation of sugar transportation and metabolism., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The nucleolus is a nuclear sub-domain containing the most highly transcribed genes in the genome. Hundreds of human ribosomal RNA (rRNA) genes, located in the nucleolus, rely on constant maintenance. DNA double-strand breaks (DSBs) in rRNA genes activate the ATM kinase, repress rRNA transcription and induce nucleolar cap formation. Yet how ribosomal-DNA (rDNA) lesions are detected and processed remains elusive. Here, we use CRISPR/Cas9-mediated induction of DSBs and report a chromatin response unique to rDNA depending on ATM-phosphorylation of the nucleolar protein TCOF1 and recruitment of the MRE11-RAD50-NBS1 (MRN) complex via the NBS1-subunit. NBS1- and MRE11-depleted cells fail to suppress rRNA transcription and to translocate rDNA into nucleolar caps. Furthermore, the DNA damage response (DDR) kinase ATR operates downstream of the ATM-TCOF1-MRN interplay and is required to fully suppress rRNA transcription and complete DSB-induced nucleolar restructuring. Unexpectedly, we find that DSBs in rDNA neither activate checkpoint kinases CHK1/CHK2 nor halt cell-cycle progression, yet the nucleolar-DDR protects against genomic aberrations and cell death. Our data highlight the concept of a specialized nucleolar DNA damage response (n-DDR) with a distinct protein composition, spatial organization and checkpoint communication. The n-DDR maintains integrity of ribosomal RNA genes, with implications for cell physiology and disease., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ionizing radiation (IR) causes DNA double-strand breaks (DSBs) and activates a versatile cellular response regulating DNA repair, cell-cycle progression, transcription, DNA replication and other processes. In recent years proteomics has emerged as a powerful tool deepening our understanding of this multifaceted response. In this study we use SILAC-based proteomics to specifically investigate dynamic changes in cytoplasmic protein abundance after ionizing radiation; we present in-depth bioinformatics analysis and show that levels of proteins involved in autophagy (cathepsins and other lysosomal proteins), proteasomal degradation (Ubiquitin-related proteins), energy metabolism (mitochondrial proteins) and particularly translation (ribosomal proteins and translation factors) are regulated after cellular exposure to ionizing radiation. Downregulation of no less than 68 ribosomal proteins shows rapid changes in the translation pattern after IR. Additionally, we provide evidence of compartmental cytosol-nuclear translocation of numerous DNA damage related proteins using protein correlation profiling. In conclusion, these results highlight unexpected cytoplasmic processes actively orchestrated after genotoxic insults and protein translocation from the cytoplasm to the nucleus as a fundamental regulatory mechanism employed to aid cell survival and preservation of genome integrity.

Maintenance of cellular homeostasis is key to prevent transformation and disease. The cellular response to DNA double-strand breaks, primarily orchestrated by the ATM/ATR kinases is one of many mechanisms that serve to uphold genome stability and homeostasis. Upon detection of double-strand breaks (DSBs), several signaling cascades are activated to halt cell cycle progression and initiate repair. Furthermore, the DNA damage response (DDR) controls cellular processes such as transcription, splicing and metabolism. Recent studies have uncovered aspects of how the DDR operates within nucleoli. It appears that the DDR controls transcription in the nucleoli, not only when DNA breaks occur in the rDNA repeats, but also when a nuclear DDR is activated. In addition, we have gained first insights into how repair of DSBs is organized in the nucleolus. Collectively, these recent studies provide a more comprehensive picture of how the DDR regulates basic cellular functions to maintain cellular homeostasis. In this review we will summarize recent findings and discuss their implications for our understanding of how the DDR regulates transcription and repair in the nucleolus., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.

To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.

ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

Rate constants for transfer of naproxen and lidocaine from different oils and oil mixtures to aqueous buffer, pH 6.00, were determined using the rotating dialysis cell. Significantly different first-order rate constants related to attainment of equilibrium, k(obs), were derived depending on the type of oil/oil mixtures used in the release experiments. For the drugs a linear correlation was found between logk(obs) and the logarithm of the partition coefficient P(app): logk(obs)=-0.68 logP(app)-0.25 (k(obs) in h(-1), n=26). A linear relationship was observed between the calculated and experimentally determined P(app) values for the oil mixtures investigated. The specific rate constants, k(ow) and k(wo), related to the partition process were derived from the determined k(obs) and P(app) values. The rate constant k(ow) representing the rate of transfer of the solute from the oil phase to the aqueous buffer was shown to be strongly dependent on the partition coefficient according to the relationship: logk(ow)=-0.68 logP(app)-log(P(app)+1)-0.25 (k(ow) in h(-1), n=26). In particular, diminished release rates were seen for oil mixtures containing castor oil most likely afforded by hydrogen bonding between the solute and the hydroxy groups of the latter vegetable oil. In this study it has been possible to alter P(app) for a specific compound up to a factor of 10 by variation of the composition of the oil vehicle. Such a span of P(app) values results in in vitro release rates differing a factor of 37. Thus, by proper design of the oil vehicle composition it should be possible to modify the release rate for a specific compound within certain limits.

The rate constants for transfer of model compounds (naproxen and lidocaine) from oily vehicle (Viscoleo) to aqueous buffer phases were determined by use of the rotating dialysis cell. Release studies were done for the partly ionized compounds at several pH values. A correlation between the overall first-order rate constant related to attainment of equilibrium, k(obs), and the pH-dependent distribution coefficient, D, determined between oil vehicle and aqueous buffer was established according to the equation: logk(obs)=-0.71 logD-0.22 (k(obs) in h(-1)). Based on this correlation it was suggested that the rate constant of a weak electrolyte at a specified D value could be considered equal to the k(obs) value for a non-electrolyte possessing a partition coefficient, P(app), the magnitude of which was equal to D. Specific rate constants k(ow) and k(wo) were calculated from the overall rate constant and the pH-dependent distribution coefficient. The rate constant representing the transport from oily vehicle to aqueous phase, k(ow), was found to be significantly influenced by the magnitude of the partition coefficient P(app) according to: logk(ow)=-0.71 logP(app)-log(P(app)+1)-0.22 (k(ow) in h(-1)).

One approach to increase drug stability and to facilitate oral absorption of low bioavailability drugs may be to design oligopeptide ester prodrugs which are stable in the gastrointestinal tract, are transported via the oligopeptide transporter, and finally release the parent drug molecule into the blood circulation and/or by its site of action. In these kinds of prodrugs the ester linkage may be broken by pH dependent and/or enzyme catalyzed hydrolysis. The objective of the present study was to investigate the degradation mechanism and rate of the model compounds Glu(OBzl)-Sar, D-Glu(OBzl)-Ala and Asp(OBzl)-Sar in aqueous solution and in relevant biological media and to compare these results with those of our previous study of D-Asp(OBzl)-Ala. Furthermore, the resulting aqueous stability and in vitro metabolism data are related to our previous affinity data to evaluate if Glu-Sar, D-Glu-Ala, and Asp-Sar have potential as pro-moieties in these kinds of prodrugs. The degradation rates follow first-order kinetics, show maximun stability at pH 4-5 with maximum half-lives for Asp(OBzl)-Sar, Glu(OBzl)-Sar, and D-Glu(OBzl)-Ala of 115 h, 30 days and 152 days, respectively. The stability was dependent on buffer concentration, temperature, pH, and ionic strength. In biological media such as 80% human plasma, human gastric juice and intestinal fluid, and 10% rat jejunal homogenate at 37 degrees C, the half-lives were greater than 1 h except for the hydrolysis of Glu(OBzl)-Sar in 10% rat jejunal homogenate, where the half-life was approximately 16 min. All the stabilized dipeptides may have potential as drug carriers targeting hPepT1.

Don H. Larsen (Brigham Young University, Provo, Utah), and R. L. Dimmick. Attachment and growth of bacteria on surfaces of continuous-culture vessels. J. Bacteriol. 88:1380-1387. 1964.-Initial attempts to induce synchrony in a continuous culture of Serratia marcescens by alternating growth temperatures produced fluctuations in the population of a magnitude and at a density higher than predicted by theory. Without temperature change, the density in the 14-ml volume changed with dilution rate, but the total output of cells per hour remained constant, even at dilution rates greater than critical. When glass wool was added to the culture vessel, the total output per hour increased 30-fold. Nonlethal ultrasonic agitation applied to the vessel reduced the population density in continuous culture under both a static and a cyclic temperature program. The decrease in population density, when the washout rate was momentarily increased about tenfold, was less than theoretically predicted, and the subsequent rapid rise, when flow was terminated, indicated the presence of a reservoir of cells on the walls of the vessel continually discharging their progeny into the medium. Several genera were examined in the latter manner; it is estimated that in some cases as many as 90% (S. marcescens, Escherichia coli), and in others (Bacillus spp.) possibly none, of the cells in suspension arose from wall inhabitants. Growth of bacteria on the walls of continuous-culture vessels can significantly influence the population density and, hence, the kinetics of continuous growth.