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Reactive oxygen species are a constant threat to DNA as they modify bases with the risk of disrupting genome function, inducing genome instability and mutation. Such risks are due to ...primary oxidative DNA damage and also mediated by the repair process. This leads to a delicate decision process for the cell as to whether to repair a damaged base at a specific genomic location or better leave it unrepaired. Persistent DNA damage can disrupt genome function, but on the other hand it can also contribute to gene regulation by serving as an epigenetic mark. When such processes are out of balance, pathophysiological conditions could get accelerated, because oxidative DNA damage and resulting mutagenic processes are tightly linked to ageing, inflammation, and the development of multiple age-related diseases, such as cancer and neurodegenerative disorders.
Recent technological advancements and novel data analysis strategies have revealed that oxidative DNA damage, its repair, and related mutations distribute heterogeneously over the genome at multiple levels of resolution. The involved mechanisms act in the context of genome sequence, in interaction with genome function and chromatin.
This review addresses what we currently know about the genome distribution of oxidative DNA damage, repair intermediates, and mutations. It will specifically focus on the various methodologies to measure oxidative DNA damage distribution and discuss the mechanistic conclusions derived from the different approaches. It will also address the consequences of oxidative DNA damage, specifically how it gives rise to mutations, genome instability, and how it can act as an epigenetic mark.
The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel ...profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.
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•The outcome of CRISPR-mediated editing can be predicted•Not all target sites are edited in a predictable manner•The precision of DNA editing is mainly determined by the fourth nucleotide upstream of the PAM site•Chromatin states affect editing of imprecise, but not precise, target sites
Chakrabarti, Henser-Brownhill, Monserrat et al. show that the genome-editing outcome can be predicted based on simple rules that mainly depend on the target site sequence. Since editing precision varies considerably across sites, careful selection of a predictable target is critical to induce a desired modification in a cell-type-independent manner.
DNA is subject to constant chemical modification and damage, which eventually results in variable mutation rates throughout the genome. Although detailed molecular mechanisms of DNA damage and repair ...are well understood, damage impact and execution of repair across a genome remain poorly defined.
To bridge the gap between our understanding of DNA repair and mutation distributions, we developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250-bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. Unlike single nucleotide variants (SNVs) in cancers, damage burden correlates with marks for open chromatin notably H3K9ac and H3K4me2. Apurinic sites and oxidative damage are also highly enriched in transposable elements and other repetitive sequences. In contrast, we observe a reduction at chromatin loop anchors with increased damage load towards inactive compartments. Less damage is found at promoters, exons, and termination sites, but not introns, in a seemingly transcription-independent but GC content-dependent manner. Leveraging cancer genomic data, we also find locally reduced SNV rates in promoters, coding sequence, and other functional elements.
Our study reveals that oxidative DNA damage accumulation and repair differ strongly across the genome, but culminate in a previously unappreciated mechanism that safeguards the regulatory and coding regions of genes from mutations.
DNA is constantly challenged by chemical modification and spontaneous loss of its bases, which results in apurinic sites (AP-sites). In addition to the direct route, modified bases may be converted ...into AP-sites through enzymatic removal of the base as part of the base excision repair pathway. Here we present the method AP-seq, which allows enriching and sequencing AP-sites genome-wide. Quantification of DNA recovery (AP-quant) allows for relative quantification of global AP-sites, and AP-site pulldown followed by qPCR (AP-qPCR) allows for site-specific damage assessment. Taking advantage of glycosylases that specifically excise modified bases also in vitro, this method allows not only to address the genomic distribution of AP-sites but also to detect base modifications, e.g., 8-oxo-7,8-dihydroguanine (8-oxoG). AP-quant, AP-qPCR, and AP-seq can be applied to investigate quantitatively the relative amount and genome specificity of DNA damage and repair, effects of radiation, as well as multiple other questions around AP-sites and base modifications.
p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1’s function is well established in DNA double-strand break repair, how its role in p53 signaling ...is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.
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•AHNAK is a G1-enriched interactor of 53BP1•AHNAK controls 53BP1-mediated G1-S phase transition upon DNA damage•AHNAK restrains 53BP1 oligomerization and phase separation•AHNAK balances between apoptosis and senescence in cancer and non-transformed cells
Ghodke et al. identify the large scaffolding protein AHNAK as a G1-enriched interactor of 53BP1 that ensures optimal partitioning of 53BP1 into phase-separated condensates and limits excessive interaction with p53, which would otherwise lead to apoptosis in cancer cells and senescence in non-transformed cells.
Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we ...performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.
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•SARS-CoV2 infection elicits dynamic changes of circulating cells in the blood•Severe COVID-19 is characterized by increased metabolically active plasmablasts•Elevation of IFN-activated megakaryocytes and erythroid cells in severe COVID-19•Cell-type-specific expression signatures are associated with a fatal COVID-19 outcome
Bernardes et al. explore COVID-19 disease trajectories by performing longitudinal multi-omics analyses in peripheral blood samples from hospitalized patients. The analyses identify increased numbers of plasmablasts, interferon-activated megakaryocytes, and erythroid cells as hallmarks of severe disease and define molecular signatures linked to a fatal COVID-19 disease outcome.
Regulator of telomere length 1 (RTEL1) is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1-deficient cells remains ...unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1−/− cells possess G-quadruplex (G4)-DNA-forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine). Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1−/− cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1−/− and Rtel1PIP_box knockin cells and in wild-type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication.
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•Rtel1 loss and G4 stabilization cause overlapping transcriptional changes•Rtel1 loss and G4 stabilization cause transcription-replication conflicts•Replication stress caused by Rtel1 loss or RTEL1/PCNA mutation is R-loop dependent•G4 stabilization causes replication stress that is R-loop dependent
Kotsantis et al. report that loss of the helicase RTEL1 leads to extensive transcriptional changes that overlap with those caused by G4 stabilization. Genome-wide replication stress caused by RTEL1 loss, RTEL1/PCNA mutation, and G4 stabilization is associated with R-loop-dependent transcription-replication conflicts.
In Germany, Eastern regions had a mild first wave of coronavirus disease 2019 (COVID-19) from March to May 2020, but were badly hit by a second wave later in autumn and winter. It is unknown how the ...second wave was initiated and developed in Eastern Germany where the number of COVID-19 cases was close to zero in June and July 2020. We used genomic epidemiology to investigate the dynamic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage development across the first and second waves in Eastern Germany. With detailed phylogenetic analyses we could show that SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with several new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August and October 2020. The results indicate that the major driving force behind the second wave was the introduction of new variants.
Abstract Epigenetic mechanisms synergize with genetic alterations in modulating gene expression patterns in cancer cells. While epigenetic alterations are reversible genetic modifications are not. ...This has raised the attention of many groups to focus on a better understanding of the molecular mechanisms that underlie the establishment of altered DNA methylation, histone modifications patterns and miRNA expression. The improved understanding of these mechanisms we will in turn allow us to improve the strategies that can be used for epigenetic therapies. In this review we will discuss and summarize briefly our current knowledge of epigenetic alterations in leukemias and will turn our attention to a concrete example of epigenetic deregulation of CCAAT/enhancer-binding protein alpha ( C/EBPα ), a key regulator for granulocytic differentiation of common myeloid progenitor cells in order to highlight the cooperativity of genetic and epigenetic mechanisms acting on this gene during the process of leukemogenesis.
For optimal pancreatic cancer treatment, early and accurate diagnosis is vital. Blood-derived biomarkers and genetic predispositions can contribute to early diagnosis, but they often have limited ...accuracy or applicability. Here, we seek to exploit the synergy between them by combining the biomarker CA19-9 with RNA-based variants. We use deep sequencing and deep learning to improve differentiating pancreatic cancer and chronic pancreatitis. We obtained samples of nucleated cells found in peripheral blood from 268 patients suffering from resectable, non-resectable pancreatic cancer, and chronic pancreatitis. We sequenced RNA with high coverage and obtained millions of variants. The high-quality variants served as input together with CA19-9 values to deep learning models. Our model achieved an area under the curve (AUC) of 96% in differentiating resectable cancer from pancreatitis using a test cohort. Moreover, we identified variants to estimate survival in resectable cancer. We show that the blood transcriptome harbours variants, which can substantially improve noninvasive clinical diagnosis.
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