Helicases in R-loop Formation and Resolution.

With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA-DNA hybrid and a displaced single-strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA-DNA hybrids. Additionally, certain helicases have strand-annealing activity. Thus, helicases possess unique positions for R-loop biogenesis: they utilize their strand-annealing activity to promote the hybridization of RNA to DNA, leading to the formation of R-loops; conversely, they utilize their unwinding activity to separate RNA-DNA hybrids and resolve R-loops. Indeed, numerous helicases such as senataxin (SETX), Aquarius (AQR), WRN, BLM, RTEL1, PIF1, FANCM, ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG, and several DEAD/H-box proteins are reported to resolve R-loops; while other helicases, such as Cas3 and UPF1, are reported to stimulate R-loop formation. Moreover, helicases like DDX1, DDX17, and DHX9 have been identified in both R-loop formation and resolution. In this review, we will summarize the latest understandings regarding the roles of helicases in R-loop metabolism. Additionally, we will highlight challenges associated with drug discovery in the context of targeting these R-loop helicases.

Adiponectin. rs266729 Polymorphism and Nicotine Dependence Interaction: Genetic Investigations on the Anxiety Susceptibility.

BACKGROUND AND AIMS: Nicotine dependence (ND)-induced anxiety might be modulated by genetic polymorphisms. The gene-by-environment interaction can be fitted into the diathesis-stress and differential susceptibility models. Nevertheless, knowledge of the interaction between adiponectin (ADPN) polymorphisms and ND on the incident mental disorder is currently scarce. This study aims to understand the role of ADPN rs266729 on anxiety in patients with ND while elucidating the psychology model and the various reactions across genotypes. METHODS: We included 315 Chinese males with confirmed ND, measured using the Fagerstrom test for nicotine dependence (FTND). Anxiety was assessed using the Self-rating Anxiety Scale. Genomic DNA was extracted and genotyped from peripheral blood. Hierarchical regression models were used to test the interactions. RESULTS: There was a significant interaction between ADPN rs266729 and ND (ß = -0.19, p < 0.05). The CC homozygote was more likely to be affected by ND-induced anxiety (ß = 0.14, t = 4.43, p < 0.01). Re-parameterized regression models revealed that the interaction between ADPN rs266729 and ND could fit the strong differential susceptibility model (R2 = 0.05, p < 0.001). CONCLUSIONS: ADPN rs266729 was correlated with susceptibility to anxiety symptoms among male adults with ND and could fit the differential susceptibility model. The CC homozygote of rs266729 was a plasticity factor that increased anxiety symptoms in individuals with ND.

Metal ions in cerebrospinal fluid: Associations with anxiety, depression, and insomnia among cigarette smokers.

OBJECTIVE: The study aimed to investigate the relationship between cerebrospinal fluid (CSF) metal ions and anxiety, depression, and insomnia among cigarette smokers. METHODS: We measured CSF levels of various metal ions from 178 Chinese male subjects. Apart from sociodemographic and clinical characteristics data, the Fagerstrom Test for Nicotine Dependence (FTND), Beck Depression Inventory (BDI), Self-Rating Anxiety Scale (SAS), and Pittsburgh Sleep Quality Index (PSQI) were applied. RESULTS: BDI and PSQI scores (all p < 0.001) were significantly higher in active smokers than nonsmokers. Active smokers have significantly higher CSF levels of magnesium, zinc, iron, lead, lithium, and aluminum (all p ≤ 0.002). Some metal ions, including zinc, iron, lead, and aluminum, were found to have a significant correlation with BDI scores, whereas metal ions, including zinc and lead, were found to have a significant correlation with PSQI scores in the general group. More interesting, mediation analysis showed that aluminum mediated the relationship between smoking and depression. CONCLUSIONS: Cigarette smoking was indeed associated with depression and insomnia. Active smokers had significantly higher CSF levels of magnesium, zinc, iron, lead, lithium, and aluminum. Furthermore, CSF aluminum played a mediating role in the relationship between smoking and depression, which further confirmed its neurotoxicity.

DDX41 is required for cGAS-STING activation against DNA virus infection.

Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.

Association of Acute Postoperative Pain and Cigarette Smoking With Cerebrospinal Fluid Levels of Beta-Endorphin and Substance P.

Objectives: Cigarette smoking is associated with postoperative pain perception, which might be mediated by beta-endorphin and substance P. These effects on postoperative pain perception have never been investigated in human cerebrospinal fluid (CSF), which reflects biochemical alterations in the brain. Therefore, we investigated the associations among cigarette smoking, postoperative pain, and levels of beta-endorphin and substance P in human CSF. Methods: We recruited 160 Chinese men (80 active smokers and 80 nonsmokers) who underwent lumbar puncture before anterior cruciate ligament reconstruction, and 5-ml CSF samples were collected. Pain visual analog scale (VAS) scores, post-anesthetic recovery duration (PARD), and smoking variables were obtained. CSF levels of beta-endorphin and substance P were measured. Results: Compared to non-smokers, active smokers had significantly higher pain VAS (2.40 ± 0.67 vs. 1.70 ± 0.86, p < 0.001) and PARD scores (9.13 ± 2.11 vs. 7.27 ± 1.35, p = 0.001), lower CSF beta-endorphin (33.76 ± 1.77 vs. 35.66 ± 2.20, p = 0.001) and higher CSF substance P (2,124.46 ± 217.34 vs. 1,817.65 ± 302.14, p < 0.001) levels. Pain VAS scores correlated with PARD in active smokers (r = 0.443, p = 0.001). Conclusions: Cigarette smoking is associated with increased postoperative pain intensity, shown by delayed pain perception, higher pain VAS scores, and lower beta-endorphin and higher substance P levels in the CSF of active smokers. The more extended postoperative pain perception is delayed, the more pain intensity increases.

The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase.

The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation-seq, and cross-linking immunoprecipitation-seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.