Explore the vast range of titles available.

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors. PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.

DNA damage is a constant source of stress challenging genomic integrity. To ensure faithful duplication of our genomes, mechanisms have evolved to deal with damage encountered during replication. One such mechanism is referred to as DNA damage tolerance (DDT). DDT allows for replication to continue in the presence of a DNA lesion by promoting damage bypass. Two major DDT pathways exist: error-prone translesion synthesis (TLS) and error-free template switching (TS). TLS recruits low-fidelity DNA polymerases to directly replicate across the damaged template, whereas TS uses the nascent sister chromatid as a template for bypass. Both pathways must be tightly controlled to prevent the accumulation of mutations that can occur from the dysregulation of DDT proteins. A key regulator of error-prone versus error-free DDT is the replication clamp, proliferating cell nuclear antigen (PCNA). Post-translational modifications (PTMs) of PCNA, mainly by ubiquitin and SUMO (small ubiquitin-like modifier), play a critical role in DDT. In this review, we will discuss the different types of PTMs of PCNA and how they regulate DDT in response to replication stress. We will also cover the roles of PCNA PTMs in lagging strand synthesis, meiotic recombination, as well as somatic hypermutation and class switch recombination.

RAD18 is a conserved eukaryotic E3 ubiquitin ligase that promotes genome stability through multiple pathways. One of these is gap-filling DNA synthesis at active replication forks and in post-replicative DNA. RAD18 also regulates homologous recombination (HR) repair of DNA breaks; however, the current literature describing the contribution of RAD18 to HR in mammalian systems has not reached a consensus. To investigate this, we examined three independent RAD18-null human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e., HCT116 mutants were hyper-recombinogenic (hyper-rec). Interestingly, these phenotypes were linked to RAD18’s role in PCNA K164 ubiquitination, as HCT116 PCNAK164R/+ mutants were also hyper-rec, consistent with previous studies in rad18−/− and pcnaK164R avian DT40 cells. Importantly, the knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. We propose that the hierarchy of post-replicative repair and HR, intrinsic to each cell type, dictates whether RAD18 is required for suppression of hyper-recombination and that this function is linked to PCNA K164 ubiquitination.

Resveratrol may be a powerful way of protecting the brain against a wide variety of stress and injury. Recently, it has been proposed that resveratrol not only reduces brain injury but also promotes recovery after stroke. But the underlying mechanisms are unclear. Here, we tested the hypothesis that resveratrol promotes angiogenesis in cerebral endothelial cells and dissected the signaling pathways involved. Treatment of cerebral endothelial cells with resveratrol promoted proliferation, migration, and tube formation in Matrigel assays. Consistent with these pro-angiogenic responses, resveratrol altered endothelial morphology resulting in cytoskeletal rearrangements of β-catenin and VE-cadherin. These effects of resveratrol were accompanied by activation of phosphoinositide 3 kinase (PI3-K)/Akt and Mitogen-Activated Protein Kinase (MAPK)/ERK signaling pathways that led to endothelial nitric oxide synthase upregulation and increased nitric oxide (NO) levels. Subsequently, elevated NO signaling increased vascular endothelial growth factor and matrix metalloproteinase levels. Sequential blockade of these signaling steps prevented resveratrol-induced angiogenesis in cerebral endothelial cells. These findings provide a mechanistic basis for the potential use of resveratrol as a candidate therapy to promote angiogenesis and neurovascular recovery after stroke.

Blood–brain barrier (BBB) disruption occurs with a high incidence after traumatic brain injury, and is an important contributor to many pathological processes, including brain edema, inflammation, and neuronal cell death. Therefore, BBB integrity is an important potential therapeutic target in the treatment of the acute phase of brain trauma. In this short communication, we report our data showing that neuregulin-1 (NRG1), a growth factor with diverse functions in the central nervous system (CNS), ameliorates pathological increases in endothelial permeability and in BBB permeability in experimental models of injury. For in vitro studies, rat brain endothelial cells were incubated with the inflammatory cytokine IL-1β, which caused an increase in permeability of the cell layer. Co-incubation with NRG1 ameliorated this permeability increase. For in vivo studies, C57Bl/6 mice were subjected to controlled cortical impact (CCI) under anesthesia, and BBB permeability was assessed by measuring the amount of Evans blue dye extravasation at 2 h. NRG1 administered by tail-vein injection 10 min after CCI resulted in a decrease in Evans blue dye extravasation by 35 %. Since Evans blue extravasation may result from an increase in BBB permeability or from bleeding due to trauma, hemoglobin ELISA was also performed at the same time point. There was a trend toward lower levels of hemoglobin extravasation in the NRG1 group, but the results did not reach statistical significance. MMP-9 activity was not different between groups at 2 h. These data suggest that NRG1 has beneficial effects on endothelial permeability and BBB permeability following experimental trauma, and may have neuroprotective potential during CNS injury.

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10 : MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients. Minichromosome maintenance protein 10 (MCM10) is critical for eukaryotic DNA replication. Here, by modelling MCM10 variants in human cell lines, the authors reveal a mechanism of MCM10-associated disease, finding that loss of MCM10 function constrains telomerase activity.

Genome integrity relies on a robust DNA replication program to ensure faithful duplication of genetic material, free from sequence mutations, deletions or rearrangements. There is an estimated 10 quadrillion (1x1016) cell divisions that occur in the average lifetime of a human being (Weinberg 2014). Thus, cells rely on a global DNA damage response (DDR) network to sense and repair errors that occur during replication to prevent the perpetuation of mutations (Ciccia and Elledge 2010). Although the DDR is highly efficient, some errors may escape repair and interfere with the progression of replication forks. In this scenario, cells utilize DNA damage tolerance (DDT) pathways to bypass errors/lesions encountered during replication and promote replication fork restart (Friedberg 2005, Chang and Cimprich 2009, Ghosal and Chen 2013). A major regulator of DDT pathways is proliferating cell nuclear antigen (PCNA) (Hoege et al. 2002). Ubiquitin modification at the conserved lysine residue 164 (K164) is crucial to DDT pathway choice – mono-ubiquitination activates error-prone translesion synthesis (TLS), while poly-ubiquitination activates error-free template switching (TS) (Shcherbakova and Fijalkowska 2006, Lehmann et al. 2007, Branzei 2011, Sale et al. 2012). However, whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear.The ends of chromosomes, known as telomeres, are origin-poor and present multiple challenges for the replication machinery including the propensity to form guanine (G)-quadruplexes and RNA-DNA hybrids (Sfeir et al. 2009, Maestroni et al. 2017). Because telomeres are intrinsically “difficult to replicate”, these regions are particularly sensitive to replication stress (Özer and Hickson 2018). In addition to the canonical replication machinery, additional proteins are needed to properly replicate the telomeric duplex. One of these proteins, the TLS polymerase η, functions to alleviate telomeric replication stress (Pope-Varsalona et al. 2014, Garcia-Exposito et al. 2016). The recruitment of TLS polymerases, including Pol η, to DNA lesions occurs through the direct interaction with mono-ubiquitinated PCNA (Bienko et al. 2005). These observations suggest a direct role for PCNA ubiquitination in the replication of telomeres. However, several reports have suggested that TLS can operate in the absence of PCNA ubiquitination (Haracska et al. 2006, Acharya et al. 2007, Parker et al. 2007, Edmunds et al. 2008, Nikolaishvili-Feinberg et al. 2008, Hendel et al. 2011, Krijger et al. 2011), thus it is not clear whether this modification is involved in telomere maintenance.While the role of PCNA-K164 ubiquitination for normal DNA replication and DDT pathway activation has been extensively studied in model systems of yeast, chicken, and mouse, how this modification functions in maintaining human genome stability is still not understood. This thesis addresses several critical functions of K164 ubiquitination in human cells. Studies in PCNAK164R mutants reveal that PCNA ubiquitination is required for gap-filling on the lagging strand behind progressing replication forks (Thakar et al. 2020). Additionally, we provide evidence that K164 ubiquitination functions to resolve late replicating intermediates (LRIs) through mitotic DNA synthesis (MiDAS) and promote efficient origin licensing in the subsequent G1 phase. Finally, we find that post-translational modification of PCNA at K164 regulates telomere maintenance specifically in transformed cells. Together, these studies show that the functions of PCNA-K164 go well beyond progressive DNA synthesis and DDT activation and extend to MiDAS and telomere maintenance.

Diabetes elevates the risk for neurological diseases, but little is known about the underlying mechanisms. Brain-derived neurotrophic factor (BDNF) is secreted by microvascular endothelial cells (ECs) in the brain, functioning as a neuroprotectant through the activation of the neurotrophic tyrosine kinase receptor TRKB. In a rat model of streptozotocin-induced hyperglycemia, we found that endothelial activation of MMP9 altered TRKB-dependent trophic pathways by degrading TRKB in neurons. Treatment of brain microvascular ECs with advanced glycation endproducts (AGE), a metabolite commonly elevated in diabetic patients, increased MMP9 activation, similar to in vivo findings. Recombinant human MMP9 degraded the TRKB ectodomain in primary neuronal cultures, suggesting that TRKB could be a substrate for MMP9 proteolysis. Consequently, AGE-conditioned endothelial media with elevated MMP9 activity degraded the TRKB ectodomain and simultaneously disrupted the ability of endothelium to protect neurons against hypoxic injury. Our findings demonstrate that neuronal TRKB trophic function is ablated by MMP9-mediated degradation in the diabetic brain, disrupting cerebrovascular trophic coupling and leaving the brain vulnerable to injury.

Neuroinflammation contributes to the pathophysiology of diverse diseases including stroke, traumatic brain injury, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, resulting in neurodegeneration and loss of neurological function. The response of the microvascular endothelium often contributes to neuroinflammation. One such response is the upregulation of endothelial adhesion molecules which facilitate neutrophil adhesion to the endothelium and their migration from blood to tissue. Neuregulin-1 (NRG1) is an endogenous growth factor which has been reported to have anti-inflammatory effects in experimental stroke models. We hypothesized that NRG1 would decrease the endothelial response to inflammation and result in a decrease in neutrophil adhesion to endothelial cells. We tested this hypothesis in an in vitro model of cytokine-induced endothelial injury, in which human brain microvascular endothelial cells (BMECs) were treated with IL-1β, along with co-incubation with vehicle or NRG1-β. Outcome measures included protein levels of endothelial ICAM-1, VCAM-1, and E-selectin, as well as the number of neutrophils that adhere to the endothelial monolayer. Our data show that NRG1-β decreased the levels of VCAM-1, E-selectin, and neutrophil adhesion to brain microvascular endothelial cells activated by IL1-β. These findings open new possibilities for investigating NRG1 in neuroprotective strategies in brain injury.

Perturbations in blood vessels play a critical role in the pathophysiology of brain injury and neurodegeneration. Here, we use a systematic genome-wide transcriptome screening approach to investigate the vasculome after brain trauma in mice. Mice were subjected to controlled cortical impact and brains were extracted for analysis at 24 h post-injury. The core of the traumatic lesion was removed and then cortical microvesels were isolated from nondirectly damaged ipsilateral cortex. Compared to contralateral cortex and normal cortex from sham-operated mice, we identified a wide spectrum of responses in the vasculome after trauma. Up-regulated pathways included those involved in regulation of inflammation and extracellular matrix processes. Decreased pathways included those involved in regulation of metabolism, mitochondrial function, and transport systems. These findings suggest that microvascular perturbations can be widespread and not necessarily localized to core areas of direct injury per se and may further provide a broader gene network context for existing knowledge regarding inflammation, metabolism, and blood–brain barrier alterations after brain trauma. Further efforts are warranted to map the vasculome with higher spatial and temporal resolution from acute to delayed phase post-trauma. Investigating the widespread network responses in the vasculome may reveal potential mechanisms, therapeutic targets, and biomarkers for traumatic brain injury.