Unscheduled homologous recombination (HR) can lead to genomic instability, which greatly increases the threat of neoplastic transformation in humans. The F-box DNA helicase 1 (FBH1) is a 3'-5' DNA ...helicase with a putative function as a negative regulator of HR. It is the only known DNA helicase to contain an F-box, suggesting that one of its functions is to act as a ubiquitin ligase as part of an SCF (SKP1, CUL1 and F-box) complex. Here we report that the central player in HR, RAD51, is ubiquitylated by the SCF(FBH1) complex. Expression of an ubiquitylation-resistant form of RAD51 in human cells leads to hyperrecombination, as well as several phenotypes indicative of an altered response to DNA replication stress. These effects are likely to be mediated by the enhanced nuclear matrix association of the ubiquitylation-resistant RAD51. These data are consistent with FBH1 acting as a negative regulator of RAD51 function in human cells.
Genome instability and cell cycle dysregulation are commonly associated with cancer. DNA replication stress driven by oncogene activation during tumorigenesis is now well established as a source of ...genome instability. Replication stress generates DNA damage not only during S phase, but also in the subsequent mitosis, where it impacts adversely on chromosome segregation. Some regions of the genome seem particularly sensitive to replication stress-induced instability; most notably, chromosome fragile sites. In this article, we review some of the important issues that have emerged in recent years concerning DNA replication stress and fragile site expression, as well as how chromosome instability is minimized by a family of ring-shaped protein complexes known as SMC proteins. Understanding how replication stress impacts on S phase and mitosis in cancer should provide opportunities for the development of novel and tumour-specific treatments.
Introduction
Green tea extract (GTE) alleviated ocular inflammations in endotoxin-induced uveitis (EIU) rat model induced by lipopolysaccharide (LPS) but the underlying mechanism is unclear.
...Objectives
To investigate the systematic and local mechanisms of the alleviation by untargeted metabolomics using liquid chromatography-tandem mass spectrometry
Methods
Sprague-Dawley rats were divided into control group, LPS treatment group, and LPS treatment group treated with GTE two hours after LPS injection. The eyes were monitored by slip lamp and electroretinography examination after 24 hours. The plasma and retina were collected for metabolomics analysis
Results
In LPS treated rats, the iris showed hyperemia. Plasma prostaglandins, arachidonic acids, corticosteroid metabolites, and bile acid metabolites increased. In the retina, histamine antagonists, corticosteroids, membrane phospholipids, free antioxidants, and sugars also increased but fatty acid metabolites, N-acetylglucosamine-6-sulphate, pyrocatechol, and adipic acid decreased. After GTE treatment, the a- and b- waves of electroretinography increased by 13%. Plasma phosphorylcholine lipids increased but plasma prostaglandin E1, cholanic metabolites, and glutarylglycine decreased. In the retina, tetranor-PGAM, pantothenic derivatives, 2-ethylacylcarinitine, and kynuramine levels decreased but anti-oxidative seleno-peptide level increased. Only phospholipids, fatty acids, and arachidonic acid metabolites in plasma and in the retina had significant correlation (p < 0.05, r > 0.4 or r < -0.4).
Conclusions
The results showed GTE indirectly induced systemic phosphorylcholine lipids to suppress inflammatory responses, hepatic damage, and respiratory mitochondrial stress in EIU rats induced by LPS. Phospholipids may be a therapeutic target of GTE for anterior chamber inflammation
In light of the high incidence and mortality rates of cancer, early and accurate diagnosis is an important priority for assigning optimal treatment for each individual with suspected illness. ...Biomarkers are crucial in the screening of patients with a high risk of developing cancer, diagnosing patients with suspicious tumours at the earliest possible stage, establishing an accurate prognosis, and predicting and monitoring the response to specific therapies. Epigenetic alterations are innovative biomarkers for cancer, due to their stability, frequency, and noninvasive accessibility in bodily fluids. Epigenetic modifications are also reversible and potentially useful as therapeutic targets. Despite this, there is still a lack of accurate biomarkers for the conclusive diagnosis of most cancer types; thus, there is a strong need for continued investigation to expand this area of research. In this review, we summarise current knowledge on methylated DNA and its implications in cancer to explore its potential as an epigenetic biomarker to be translated for clinical application. We propose that the identification of biomarkers with higher accuracy and more effective detection methods will enable improved clinical management of patients and the intervention at early-stage disease.
Inactivation of the retinoblastoma tumor suppressor gene (
) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand ...breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.
Inflammation is in a wide spectrum of retinal diseases, causing irreversible blindness and visual impairment. We have previously demonstrated that Green Tea Extract (GTE) is a potent ...anti-inflammatory agent for anterior uveitis. Here we investigated the anti-inflammatory effect of GTE on lipopolysaccharides (LPS)-induced retinal inflammation in rats and explored the underlying mechanism. Adult rats were injected with LPS and GTE was administered intra-gastrically at 2, 8, 26 and 32 hours post-injection. Staining of whole-mount retina showed that the number of activated microglia cells was significantly increased at 48 hours post-injection, which was suppressed after GTE treatment in a dose-dependent manner. Activation of astrocytes and Müller glia in the retina was also suppressed after GTE treatment. Meanwhile, GTE reduced the expression of pro-inflammatory cytokines including IL-1β, TNF-α and IL-6 in retina and vitreous humor. These anti-inflammatory effects were associated with a reduced phosphorylation of STAT3 and NF-κB in the retina. Furthermore, the surface receptor of EGCG, 67LR, was localized on the neurons and glia in the retina. These findings demonstrate that GTE is an effective agent in suppressing LPS-induced retinal inflammation, probably through its potent anti-oxidative property and a receptor-mediated action on transcription factors that regulate production of pro-inflammatory cytokines.
Autosomal recessive congenital hereditary endothelial dystrophy (CHED2) may be misdiagnosed as primary congenital glaucoma (PCG) due to similar clinical phenotypes during early infancy. In this ...study, we identified a family with CHED2, which was previously misdiagnosed as having PCG, and followed up for 9 years. Linkage analysis was first completed in eight PCG-affected families, followed by whole-exome sequencing (WES) in family PKGM3. The following in silico tools were used to predict the pathogenic effects of identified variants: I-Mutant 2.0, SIFT, Polyphen-2, PROVEAN, mutation taster and PhD-SNP. After detecting an
variant in one family, detailed ophthalmic examinations were performed again to confirm the diagnosis. Six out of eight families had
gene variants responsible for PCG. However, in family PKGM3, no variants in the known PCG genes were identified. WES identified a homozygous missense variant c.2024A>C, p.(Glu675Ala) in
. Based on the WES findings, the affected individuals underwent detailed ophthalmic examinations and were re-diagnosed with CHED2 leading to secondary glaucoma. Our results expand the genetic spectrum of CHED2. This is the first report from Pakistan of a Glu675Ala variant with CHED2 leading to secondary glaucoma. The p.Glu675Ala variant is likely a founder mutation in the Pakistani population. Our findings suggest that genome-wide neonatal screening is worthwhile to avoid the misdiagnosis of phenotypically similar diseases such as CHED2 and PCG.
Genetic associations for keratoconus could be useful for understanding disease pathogenesis and discovering biomarkers for early detection of the disease. We conducted a systematic review and ...meta-analysis to summarize all reported genetic associations for the disease. We searched in the MEDLINE, Embase, Web of Science, and HuGENET databases for genetic studies of keratoconus published from 1950 to June 2016. The summary odds ratio and 95% confidence intervals of all polymorphisms were estimated using the random-effect model. Among 639 reports that were retrieved, 24 fulfilled required criteria as eligible studies for meta-analysis, involving a total of 53 polymorphisms in 28 genes/loci. Results of our meta-analysis lead to the prioritization of 8 single-nucleotide polymorphisms (SNPs) in 6 genes/loci for keratoconus in Whites. Of them 5 genes/loci were originally detected in genome-wide association studies, including FOXO1 (rs2721051, P = 5.6 × 10
), RXRA-COL5A1 (rs1536482, P = 2.5 × 10
), FNDC3B (rs4894535, P = 1.4 × 10
), IMMP2L (rs757219, P = 6.1 × 10
; rs214884, P = 2.3 × 10
), and BANP-ZNF469 (rs9938149, P = 1.3 × 10
). The gene COL4A4 (rs2229813, P = 1.3 × 10
; rs2228557, P = 4.5 × 10
) was identified in previous candidate gene studies. We also found SNPs in 10 genes/loci that had a summary P value < 0.05. Sensitivity analysis indicated that the results were robust. Replication studies and understanding the roles of these genes in keratoconus are warranted.
Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a ...repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.
Background: Homologous recombination is regulated both positively and negatively in eukaryotic cells to suppress genomic instability.
Results: FBH1 can disrupt RAD51 filaments in vitro and suppresses formation of spontaneous RAD51 foci in mammalian cells. In cells defective for FBH1, hyper-recombination is observed.
Conclusion: FBH1 is a negative regulator of homologous recombination.
Significance: RAD51 activity must be carefully controlled to preserve genomic integrity.
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