DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

UAP56/DDX39B is a major cotranscriptional RNA-DNA helicase that unwinds harmful R loops genome-wide.

Nonscheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R-loop accumulation. One mechanism relies on the conserved THO complex in association with cotranscriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R-loop removal. We show that UAP56 depletion causes R-loop accumulation, R-loop-mediated genome instability, and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and show that its overexpression suppresses R loops and genome instability induced by depleting five different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful cotranscriptional RNA structures that otherwise would block transcription and replication.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Correlates of protection and determinants of SARS-CoV-2 breakthrough infections 1 year after third dose vaccination.

BACKGROUND: The emergence of new SARS-CoV-2 variants and the waning of immunity raise concerns about vaccine effectiveness and protection against COVID-19. While antibody response has been shown to correlate with the risk of infection with the original variant and earlier variants of concern, the effectiveness of antibody-mediated protection against Omicron and the factors associated with protection remain uncertain. METHODS: We evaluated antibody responses to SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from Wuhan and variants of concern by Luminex and their role in preventing breakthrough infections 1 year after a third dose of mRNA vaccination, in a cohort of health care workers followed since the pandemic onset in Spain (N = 393). Data were analyzed in relation to COVID-19 history, demographic factors, comorbidities, vaccine doses, brand, and adverse events. RESULTS: Higher levels of anti-S IgG and IgA to Wuhan, Delta, and Omicron were associated with protection against vaccine breakthroughs (IgG against Omicron S antigen HR, 0.06, 95%CI, 0.26-0.01). Previous SARS-CoV-2 infection was positively associated with antibody levels and protection against breakthroughs, and a longer time since last infection was associated with lower protection. In addition, priming with BNT162b2 followed by mRNA-1273 booster was associated with higher antibody responses than homologous mRNA-1273 vaccination. CONCLUSIONS: Data show that IgG and IgA induced by vaccines against the original strain or by hybrid immunization are valid correlates of protection against Omicron BA.1 despite immune escape and support the benefits of heterologous vaccination regimens to enhance antibodies and the prioritization of booster vaccination in individuals without recent infections.

Extraction of Antioxidants from Brown Macroalgae Fucus spiralis.

In this study, different extraction methods and conditions were used for the extraction of antioxidants from brown macroalgae Fucus spiralis. The extraction methodologies used were ultrasound-assisted extraction (ultrasonic bath and ultrasonic probe), extraction with a vortex, extraction with an Ultra-Turrax® homogenizer, and high-pressure-assisted extraction. The extracts were analyzed for their total phenolic content (TPC) and their antioxidant activity, and evaluated through the 2,2-difenil-1-picrilhidrazil (DPPH) free radical scavenging method and ferric reducing antioxidant power (FRAP) assay. Ultrasonic probe-assisted extraction yielded the highest values of TPC (94.78-474.16 mg gallic acid equivalents/g extract). Regarding the antioxidant activity, vortex-assisted extraction gave the best DPPH results (IC50 1.89-16 µg/mL), while the highest FRAP results were obtained using the Ultra-Turrax® homogenizer (502.16-1188.81 µmol ascorbic acid equivalents/g extract). For each extraction method, response surface methodology was used to analyze the influence of the experimental conditions "extraction time" (t), "biomass/solvent ratio" (R), "solvent" (S, water % in water/ethanol mixture), and "pressure" (P) on TPC, DPPH, and FRAP of the F. spiralis extracts. In general, higher TPC content and higher antioxidant capacity (lower IC50 and higher FRAP) were obtained with higher R, t, and P, and lower S (higher ethanol %). The model regarding the combined effects of independent variables t, R, and S on the FRAP response values for vortex-assisted extractions best fitted the experimental data (R2 0.957), with optimal extraction conditions of t = 300 s, R = 50 g, and S = 25%.

TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage.

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.

The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription-replication conflicts.

FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.

Severe Acute Respiratory Syndrome Coronavirus 2 Normalized Viral Loads and Subgenomic RNA Detection as Tools for Improving Clinical Decision Making and Work Reincorporation.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) provides a highly variable cycle threshold (Ct) value that cannot distinguish viral infectivity. Subgenomic ribonucleic acid (sgRNA) has been used to monitor active replication. Given the importance of long RT-PCR positivity and the need for work reincorporation and discontinuing isolation, we studied the functionality of normalized viral loads (NVLs) for patient monitoring and sgRNA for viral infectivity detection. METHODS: The NVLs measured through the Nucleocapsid and RNA-dependent-RNA-polymerase genes and sgRNA RT-PCRs were performed in 2 consecutive swabs from 84 healthcare workers. RESULTS: The NVLs provided similar and accurate quantities of both genes of SARS-CoV-2 at 2 different timepoints of infection, overcoming Ct-value and swab collection variability. Among SARS-CoV-2-positive samples, 51.19% were sgRNA-positive in the 1st RT-PCR and 5.95% in the 2nd RT-PCR. All sgRNA-positive samples had >4 log10 RNA copies/1000 cells, whereas samples with ≤1 log10 NVLs were sgRNA-negative. Although NVLs were positive until 29 days after symptom onset, 84.1% of sgRNA-positive samples were from the first 7 days, which correlated with viral culture viability. Multivariate analyses showed that sgRNA, NVLs, and days of symptoms were significantly associated (P < .001). CONCLUSIONS: The NVLs and sgRNA are 2 rapid accessible techniques that could be easily implemented in routine hospital practice providing a useful proxy for viral infectivity and coronavirus disease 2019 patient follow-up.

SARS-CoV-2 Seroprevalence and Antibody Kinetics Among Health Care Workers in a Spanish Hospital After 3 Months of Follow-up.

BACKGROUND: At the COVID-19 spring 2020 pandemic peak in Spain, prevalence of SARS-CoV-2 infection in a cohort of 578 randomly selected health care workers (HCWs) from Hospital Clínic de Barcelona was 11.2%. METHODS: A follow-up survey 1 month later (April-May 2020) measured infection by rRT-PCR and IgM, IgA, and IgG to the receptor-binding domain of the spike protein by Luminex. Antibody kinetics, including IgG subclasses, was assessed until month 3. RESULTS: At month 1, the prevalence of infection measured by rRT-PCR and serology was 14.9% (84/565) and seroprevalence 14.5% (82/565). We found 25 (5%) new infections in 501 participants without previous evidence of infection. IgM, IgG, and IgA levels declined in 3 months (antibody decay rates 0.15 [95% CI, .11-.19], 0.66 [95% CI, .54-.82], and 0.12 [95% CI, .09-.16], respectively), and 68.33% of HCWs had seroreverted for IgM, 3.08% for IgG, and 24.29% for IgA. The most frequent subclass responses were IgG1 (highest levels) and IgG2, followed by IgG3, and only IgA1 but no IgA2 was detected. CONCLUSIONS: Continuous and improved surveillance of SARS-CoV-2 infections in HCWs remains critical, particularly in high-risk groups. The observed fast decay of IgA and IgM levels has implications for seroprevalence studies using these isotypes.

The DNA damage response acts as a safeguard against harmful DNA-RNA hybrids of different origins.

Despite playing physiological roles in specific situations, DNA-RNA hybrids threat genome integrity. To investigate how cells do counteract spontaneous DNA-RNA hybrids, here we screen an siRNA library covering 240 human DNA damage response (DDR) genes and select siRNAs causing DNA-RNA hybrid accumulation and a significant increase in hybrid-dependent DNA breakage. We identify post-replicative repair and DNA damage checkpoint factors, including those of the ATM/CHK2 and ATR/CHK1 pathways. Thus, spontaneous DNA-RNA hybrids are likely a major source of replication stress, but they can also accumulate and menace genome integrity as a consequence of unrepaired DSBs and post-replicative ssDNA gaps in normal cells. We show that DNA-RNA hybrid accumulation correlates with increased DNA damage and chromatin compaction marks. Our results suggest that different mechanisms can lead to DNA-RNA hybrids with distinct consequences for replication and DNA dynamics at each cell cycle stage and support the conclusion that DNA-RNA hybrids are a common source of spontaneous DNA damage that remains unsolved under a deficient DDR.

R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.

Study of the Seasonal Variations of the Fatty Acid Profiles of Selected Macroalgae.

Due to the high consumption of fat-rich processed foods, efforts are being done to reduce their saturated fat (SFA) contents and replace it with polyunsaturated fatty acids (PUFA), creating a necessity to find alternative PUFA sources. Macroalgae, being a promising natural source of healthy food, may be such an alternative. The fatty acid (FA) profile of Fucus spiralis, Bifurcaria bifurcata, Ulva lactuca, and Saccorhiza polyschides were determined through direct transesterification and their seasonal variation was studied. F. spiralis showed the highest FA content overall, B. bifurcata presented the higher PUFA amounts, and U. lactuca and S. polyschides the higher SFA. The production of FA was shown to be influenced by the seasons. Spring and summer seemed to induce the FA production in F. spiralis and B. bifurcata while in U. lactuca the same was verified in winter. U. lactuca presented a ω6/ω3 ratio between 0.59 and 1.38 while B. bifurcata presented a ratio around 1.31. The study on the seasonal variations of the macroalgal FA profile can be helpful to understand the best season to yield FA of interest, such as ALA, EPA, and DHA. It may also provide valuable information on the best culturing conditions for the production of desired FAs.

Seasonal Evaluation of Phlorotannin-Enriched Extracts from Brown Macroalgae Fucus spiralis.

Fucus spiralis that was collected in the four seasons was submitted to an extraction with ethanol:water (crude extracts Et80), followed by a liquid-liquid fractionation with organic solvents (fraction He from n-hexane; aqueous fractions AQ1, AQ2, AQ3 and AQ4; ethyl acetate fraction EA), with the aim of obtaining phlorotannin-enriched extracts. All the extracts (Et80, He, AQ1, AQ2, AQ3, AQ4 and EA) that were obtained for the F. spiralis of the four seasons were evaluated for their antioxidant capacity and total phenolic compounds. The summer extracts presented the highest contents in polyphenols (TPC), as well as the highest ferric reducing antioxidant power (FRAP), when compared to the samples from the other seasons. The reductive percentage of the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) compound was similar between the seasons. For all the seasons, the EA extract showed the highest polyphenol content (TPC), and the highest antioxidant capacity (highest ferric reducing power (FRAP) and lowest concentration needed to reduce 50% of the DPPH compound), which is in agreement with a phlorotannin-enriched fraction. This study revealed that the polyphenol content and antioxidant power of the F. spiralis extracts are influenced by the time of harvest, as well as by the solvents used for their extraction.

BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2.

Genome instability is central to ageing, cancer and other diseases. It is not only proteins involved in DNA replication or the DNA damage response (DDR) that are important for maintaining genome integrity: from yeast to higher eukaryotes, mutations in genes involved in pre-mRNA splicing and in the biogenesis and export of messenger ribonucleoprotein (mRNP) also induce DNA damage and genome instability. This instability is frequently mediated by R-loops formed by DNA-RNA hybrids and a displaced single-stranded DNA. Here we show that the human TREX-2 complex, which is involved in mRNP biogenesis and export, prevents genome instability as determined by the accumulation of γ-H2AX (Ser-139 phosphorylated histone H2AX) and 53BP1 foci and single-cell electrophoresis in cells depleted of the TREX-2 subunits PCID2, GANP and DSS1. We show that the BRCA2 repair factor, which binds to DSS1, also associates with PCID2 in the cell. The use of an enhanced green fluorescent protein-tagged hybrid-binding domain of RNase H1 and the S9.6 antibody did not detect R-loops in TREX-2-depleted cells, but did detect the accumulation of R-loops in BRCA2-depleted cells. The results indicate that R-loops are frequently formed in cells and that BRCA2 is required for their processing. This link between BRCA2 and RNA-mediated genome instability indicates that R-loops may be a chief source of replication stress and cancer-associated instability.

Epigenetic features of human telomeres.

Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, different human cell lines might carry diverse epigenetic marks at telomeres. We have developed a reliable procedure to study the chromatin structure of human telomeres independently of subtelomeres and ITSs. This procedure is based on the statistical analysis of multiple ChIP-seq experiments. We have found that human telomeres are not enriched in the heterochromatic H3K9me3 mark in most of the common laboratory cell lines, including embryonic stem cells. Instead, they are labeled with H4K20me1 and H3K27ac, which might be established by p300. These results together with previously published data argue that subtelomeric heterochromatin might control human telomere functions. Interestingly, U2OS cells that exhibit alternative lengthening of telomeres have heterochromatic levels of H3K9me3 in their telomeres.

N-Heterocyclic Olefin Catalysis for the Ring Opening of Cyclic Amidine Compounds: A Pathway to the Synthesis of ε-Caprolactam- and γ-Lactam-Derived Amines.

For the first time, 1,2-dimethyl-3-ethylimidazolium iodide (1a) catalyzes the ring opening of the bicyclic amidine system of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or DBN (1,5-diazabicyclo[4.3.0]non-5-ene) on reaction with aldehydes. The mechanism here proposed involves an N-heterocyclic olefin (NHO) catalytic species that acts as a nucleophile to promote the cyclic amidine ring opening. The resulting ε-caprolactam- and γ-lactam-derived imines were obtained in moderate to excellent yields (28-99%) and reduced to the corresponding amines by sodium borohydride. Confirmation of the imine product was achieved via single-crystal X-ray diffraction studies.

Coordinated control of replication and transcription by a SAPK protects genomic integrity.

Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression. Dealing with high levels of transcription during replication is critical to prevent collisions between the transcription and replication pathways and avoid recombination events. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc1(3A)) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc1(3A) induces Rad53 and survival in the presence of hydroxyurea or methyl methanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops.

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

Polycomb RING1A- and RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S-phase progression.

The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress. We have used a cellular model of conditionally inactive polycomb E3 ligases (RING1A and RING1B), which monoubiquitylate lysine 119 of histone H2A (H2AK119Ub), to examine DNA replication in unperturbed cells. We identify slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with chromocenters, the sites of coalesced pericentromeric heterocromatic (PCH) domains, were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methyl-CpG binding domain protein 1. The acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1A) upregulation, could be uncoupled from a response to DNA damage. These findings link cell proliferation and the polycomb proteins RING1A and RING1B to S-phase progression through a specific function in PCH replication.