PRL1 interacts with and stabilizes RPA2A to regulate carbon deprivation-induced senescence in Arabidopsis.

Leaf senescence is a developmental program regulated by both endogenous and environmental cues. Abiotic stresses such as nutrient deprivation can induce premature leaf senescence, which profoundly impacts plant growth and crop yield. However, the molecular mechanisms underlying stress-induced senescence are not fully understood. In this work, employing a carbon deprivation (C-deprivation)-induced senescence assay in Arabidopsis seedlings, we identified PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a component of the NineTeen Complex, as a negative regulator of C-deprivation-induced senescence. Furthermore, we demonstrated that PRL1 directly interacts with the RPA2A subunit of the single-stranded DNA-binding Replication Protein A (RPA) complex. Consistently, the loss of RPA2A leads to premature senescence, while increased expression of RPA2A inhibits senescence. Moreover, overexpression of RPA2A reverses the accelerated senescence in prl1 mutants, and the interaction with PRL1 stabilizes RPA2A under C-deprivation. In summary, our findings reveal the involvement of the PRL1-RPA2A functional module in C-deprivation-induced plant senescence.

The C-terminal amino acid motifs of NS1 protein affect the replication and virulence of naturally NS-truncated H1N1 canine influenza virus.

AbstractThe vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-ß in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

Parent-of-origin-specific DNA replication timing is confined to large imprinted regions.

Genomic imprinting involves differential DNA methylation and gene expression between homologous paternal and maternal loci. It remains unclear, however, whether DNA replication also shows parent-of-origin-specific patterns at imprinted or other genomic regions. Here, we investigate genome-wide asynchronous DNA replication utilizing uniparental human embryonic stem cells containing either maternal-only (parthenogenetic) or paternal-only (androgenetic) DNA. Four clusters of imprinted genes exhibited differential replication timing based on parent of origin, while the remainder of the genome, 99.82%, showed no significant replication asynchrony between parental origins. Active alleles in imprinted gene clusters replicated earlier than their inactive counterparts. At the Prader-Willi syndrome locus, replication asynchrony spanned virtually the entirety of S phase. Replication asynchrony was carried through differentiation to neuronal precursor cells in a manner consistent with gene expression. This study establishes asynchronous DNA replication as a hallmark of large imprinted gene clusters.

Tolerating DNA damage by repriming: Gap filling in the spotlight.

Timely and accurate DNA replication is critical for safeguarding genome integrity and ensuring cell viability. Yet, this process is challenged by DNA damage blocking the progression of the replication machinery. To counteract replication fork stalling, evolutionary conserved DNA damage tolerance (DDT) mechanisms promote DNA damage bypass and fork movement. One of these mechanisms involves "skipping" DNA damage through repriming downstream of the lesion, leaving single-stranded DNA (ssDNA) gaps behind the advancing forks (also known as post-replicative gaps). In vertebrates, repriming in damaged leading templates is proposed to be mainly promoted by the primase and polymerase PRIMPOL. In this review, we discuss recent advances towards our understanding of the physiological and pathological conditions leading to repriming activation in human models, revealing a regulatory network of PRIMPOL activity. Upon repriming by PRIMPOL, post-replicative gaps formed can be filled-in by the DDT mechanisms translesion synthesis and template switching. We discuss novel findings on how these mechanisms are regulated and coordinated in time to promote gap filling. Finally, we discuss how defective gap filling and aberrant gap expansion by nucleases underlie the cytotoxicity associated with post-replicative gap accumulation. Our increasing knowledge of this repriming mechanism - from gap formation to gap filling - is revealing that targeting the last step of this pathway is a promising approach to exploit post-replicative gaps in anti-cancer therapeutic strategies.

Mechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes.

Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, inhibitors that trap Top1-ccs are used extensively in research and clinical settings to generate DNA replication stress, yet how the replisome responds upon collision with a Top1-cc remains obscure. By reconstituting collisions between budding yeast replisomes, assembled from purified proteins, and site-specific Top1-ccs, we have uncovered mechanisms underlying replication fork stalling and collapse. We find that stalled replication forks are surprisingly stable and that their stability is influenced by the template strand that Top1 is crosslinked to, the fork protection complex proteins Tof1-Csm3 (human TIMELESS-TIPIN), and the convergence of replication forks. Moreover, nascent-strand mapping and cryoelectron microscopy (cryo-EM) of stalled forks establishes replisome remodeling as a key factor in the initial response to Top1-ccs. These findings have important implications for the use of Top1 inhibitors in research and in the clinic.

Replication-deficient sendai virus expressing human norovirus capsid protein elicits robust NoV-specific antibody and T-cell responses in mice.

Human norovirus (HuNoV) is a major global cause of acute gastroenteritis, with vaccine development facing several challenges. Despite years of research, there are currently no licensed vaccines available for controlling HuNoVs. Here, we describe the construction and testing of a replication-deficient Sendai virus (SeV) vector as a potential vaccine candidate against the HuNoV GII.4 genotype. SeV was chosen as the vaccine backbone due to its non-pathogenic nature in humans, its capability for long-term antigen expression in mammalian cells, and its suitability for mucosal administration. By inserting the HuNoV GII.4 capsid gene, VP1, into the SeV genome, we generated a replication-deficient SeV (SeV/dP.VP1) vector. The resultant SeV/dP.VP1 virus were observed to successfully express the inserted NoV VP1 gene upon infection. Inoculating the vaccine into wild-type mice elicited NoV-specific IgG antibodies, along with INF-γ and IL-2-producing T cells, through both intranasal (i.n.) and intramuscular (i.m.) immunization. Furthermore, a significant level of NoV-specific IgA was detected in lung homogenates after i.n. immunization, particularly using a high dose of the viral vector. Additionally, a synergistic effect was observed with heterologous prime-boost regimens using SeV/dP.VP1 and MVA.VP1 vectors, indicating the potential for more robust immune responses when the vaccine design is optimized. Our study demonstrates the potential of a SeV vaccine candidate in eliciting a broad immune response and lays the foundation for further exploration of the SeV vector platform's potential as a HuNoV vaccine. Additionally, the results emphasize the importance of vaccine dosage and administration route, highlighting the need for tailored immunization strategies.

Multifaceted role of the DNA replication protein MCM10 in maintaining genome stability and its implication in human diseases.

MCM10 plays a vital role in genome duplication and is crucial for DNA replication initiation, elongation, and termination. It coordinates several proteins to assemble at the fork, form a functional replisome, trigger origin unwinding, and stabilize the replication bubble. MCM10 overexpression is associated with increased aggressiveness in breast, cervical, and several other cancers. Disruption of MCM10 leads to altered replication timing associated with initiation site gains and losses accompanied by genome instability. Knockdown of MCM10 affects the proliferation and migration of cancer cells, manifested by DNA damage and replication fork arrest, and has recently been shown to be associated with clinical conditions like CNKD and RCM. Loss of MCM10 function is associated with impaired telomerase activity, leading to the accumulation of abnormal replication forks and compromised telomere length. MCM10 interacts with histones, aids in nucleosome assembly, binds BRCA2 to maintain genome integrity during DNA damage, prevents lesion skipping, and inhibits PRIMPOL-mediated repriming. It also interacts with the fork reversal enzyme SMARCAL1 and inhibits fork regression. Additionally, MCM10 undergoes several post-translational modifications and contributes to transcriptional silencing by interacting with the SIR proteins. This review explores the mechanism associated with MCM10's multifaceted role in DNA replication initiation, chromatin organization, transcriptional silencing, replication stress, fork stability, telomere length maintenance, and DNA damage response. Finally, we discuss the role of MCM10 in the early detection of cancer, its prognostic significance, and its potential use in therapeutics for cancer treatment.

Heterozygous RPA2 variant as a novel genetic cause of telomere biology disorders.

Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans.

Proteomic networks and related genetic variants associated with smoking and chronic obstructive pulmonary disease.

BACKGROUND: Studies have identified individual blood biomarkers associated with chronic obstructive pulmonary disease (COPD) and related phenotypes. However, complex diseases such as COPD typically involve changes in multiple molecules with interconnections that may not be captured when considering single molecular features. METHODS: Leveraging proteomic data from 3,173 COPDGene Non-Hispanic White (NHW) and African American (AA) participants, we applied sparse multiple canonical correlation network analysis (SmCCNet) to 4,776 proteins assayed on the SomaScan v4.0 platform to derive sparse networks of proteins associated with current vs. former smoking status, airflow obstruction, and emphysema quantitated from high-resolution computed tomography scans. We then used NetSHy, a dimension reduction technique leveraging network topology, to produce summary scores of each proteomic network, referred to as NetSHy scores. We next performed a genome-wide association study (GWAS) to identify variants associated with the NetSHy scores, or network quantitative trait loci (nQTLs). Finally, we evaluated the replicability of the networks in an independent cohort, SPIROMICS. RESULTS: We identified networks of 13 to 104 proteins for each phenotype and exposure in NHW and AA, and the derived NetSHy scores significantly associated with the variable of interests. Networks included known (sRAGE, ALPP, MIP1) and novel molecules (CA10, CPB1, HIS3, PXDN) and interactions involved in COPD pathogenesis. We observed 7 nQTL loci associated with NetSHy scores, 4 of which remained after conditional analysis. Networks for smoking status and emphysema, but not airflow obstruction, demonstrated a high degree of replicability across race groups and cohorts. CONCLUSIONS: In this work, we apply state-of-the-art molecular network generation and summarization approaches to proteomic data from COPDGene participants to uncover protein networks associated with COPD phenotypes. We further identify genetic associations with networks. This work discovers protein networks containing known and novel proteins and protein interactions associated with clinically relevant COPD phenotypes across race groups and cohorts.

Exposure of Arabidopsis thaliana Mutants to Genotoxic Stress Provides New Insights for the Involvement of TDP1α and TDP1ß genes in DNA-Damage Response.

Genotoxic stress activates the DNA-damage response (DDR) signalling cascades responsible for maintaining genome integrity. Downstream DNA repair pathways include the tyrosyl-DNA phosphodiesterase 1 (TDP1) enzyme that hydrolyses the phosphodiester bond between the tyrosine of topoisomerase I (TopI) and 3'-phosphate of DNA. The plant TDP1 subfamily contains the canonical TDP1α gene and the TDP1ß gene whose functions are not fully elucidated. The current study proposes to investigate the involvement of TDP1 genes in DDR-related processes by using Arabidopsis thaliana mutants treated with genotoxic agents. The phenotypic and molecular characterization of tdp1α, tdp1ß and tdp1α/ß mutants treated with cisplatin (CIS), curcumin (CUR), NSC120686 (NSC), zeocin (ZEO), and camptothecin (CPT), evidenced that while tdp1ß was highly sensitive to CIS and CPT, tdp1α was more sensitive to NSC. Gene expression analyses showing upregulation of the TDP2 gene in the double mutant indicate the presence of compensatory mechanisms. The downregulation of POL2A gene in the tdp1ß mutant along with the upregulation of the TDP1ß gene in pol2a mutants, together with its sensitivity to replication inhibitors (CIS, CTP), point towards a function of this gene in the response to replication stress. Therefore, this study brings novel information relative to the activity of TDP1 genes in plants.

E3 ubiquitin ligase FBXO22 inhibits SARS-CoV-2 replication via promoting proteasome-dependent degradation of NSP5.

The ubiquitin-proteasome system is frequently employed to degrade viral proteins, thereby inhibiting viral replication and pathogenicity. Through an analysis of the degradation kinetics of all the SARS-CoV-2 proteins, our study revealed rapid degradation of several proteins, particularly NSP5. Additionally, we identified FBXO22, an E3 ubiquitin ligase, as the primary regulator of NSP5 ubiquitination. Moreover, we validated the interaction between FBXO22 and NSP5, demonstrating that FBXO22-mediated ubiquitination of NSP5 facilitated its recognition by the proteasome, leading to subsequent degradation. Specifically, FBXO22 catalyzed the formation of K48-linked polyubiquitin chains on NSP5 at lysine residues 5 and 90. Knockdown of FBXO22 resulted in decreased NSP5 ubiquitination levels, increased stability, and enhanced ability to evade the host innate immune response. Notably, the protein level of FBXO22 were negatively correlated with SARS-CoV-2 load, highlighting its importance in inhibiting viral replication. This study elucidates the molecular mechanism by which FBXO22 mediates the degradation of NSP5 and underscores its critical role in limiting viral replication. The identification of FBXO22 as a regulator of NSP5 stability provides new insights and potential avenues for targeting NSP5 in antiviral strategies.

Structural Insight Into the Function of DnaB Helicase in Bacterial DNA Replication.

In bacteria, chromosome replication is achieved by the coordinations of more than a dozen replisome enzymes. Replication initiation protein DnaA melts DNA duplex at replication origin (oriC) and forms a replication bubble, followed by loading of helicase DnaB with the help of loader protein DnaC. Then the DnaB helicase unwinds the dsDNA and supports the priming of DnaG and the polymerizing of DNA polymerase. The DnaB helicase functions as a platform coupling unwinding, priming, and polymerizing events. The multiple roles of DnaB helicase are underlined by its distinctive architecture and dynamics conformations. In this review, we will discuss the assembling of DnaB hexamer and the conformational changes upon binding of various partners, DnaB in states of closed dilated (CD), closed constricted (CC), closed helical (CH), and open helical (OH) are discussed. These multiple interfaces among DnaB and partners are potential targets for inhibitors design and novel peptide antibiotics development.

Getah virus Nsp3 binds G3BP to block formation of bona fide stress granules.

Stress granules (SGs) are cytoplasmic aggregates of proteins and mRNA that form in response to diverse environmental stressors, including viral infections. Several viruses possess the ability to block the formation of stress granules by targeting the SGs marker protein G3BP. However, the molecular functions and mechanisms underlying the regulation of SGs formation by Getah virus (GETV) remain unclear. In this study, we found that GETV infection triggered the formation of Nsp3-G3BP aggregates, which differed in composition from SGs. Further studies revealed that the presence of these aggregates was dependent on the activation of the PKR/eIF2α signaling pathway. Interestingly, we found that Nsp3 HVD domain blocked the formation of SGs by binding to G3BP NTF2 domain. Moreover, knockout of G3BP in NCI-H1299 cells had no effect on GETV replication, while overexpression of G3BP to form the genuine SGs significantly inhibited GETV replication. Overall, our study elucidates a novel role GETV Nsp3 to change the composition of SG as well as cellular stress response.

In vitro observation of histone-hexamer association with and dissociation from the amino-terminal region of budding yeast Mcm2, a subunit of the replicative helicase.

During DNA replication, core histones that form nucleosomes on template strands are evicted and associate with newly synthesized strands to reform nucleosomes. Mcm2, a subunit of the Mcm2-7 complex, which is a core component of the replicative helicase, interacts with histones in the amino-terminal region (Mcm2N) and is involved in the parental histone recycling to lagging strands. Herein, the interaction of Mcm2N with histones was biochemically analyzed to reveal the molecular mechanisms underlying histone recycling by Mcm2N. With the addition of Mcm2N, a histone hexamer, comprising a H3-H4 tetramer and a H2A-H2B dimer, was excised from the histone octamer to form a complex with Mcm2N. The histone hexamer, but not H3-H4 tetramer was released from Mcm2N in the presence of Nap1, a histone chaperone. FACT, another histone chaperone, stabilized Mcm2N-histone hexamer complex to protect from Nap1-dependent dissociation. This study indicates cooperative histone transfer via Mcm2N and histone chaperones.

Does Physiological Arousal Increase Social Transmission of Information? Two Replications of Berger (2011).

People share information for many reasons. For example, Berger (2011, N = 40) found that undergraduate participants manipulated to have higher physiological arousal were more likely to share a news article with others via email than people who had low arousal. Berger's research is widely cited as evidence of the causal role of arousal in sharing information and has been used to explain why information that induces high-arousal emotions is shared more than information that induces low-arousal emotions. We conducted two replications (N = 111, N = 160) of Berger's study, using the same arousal manipulation but updating the sharing measure to reflect the rise of information sharing through social media. Both studies failed to find an impact of incidental physiological arousal on undergraduate participants' willingness to share news articles on social media. Our studies cast doubt on the idea that incidental physiological arousal-in the absence of other factors-impacts people's decisions to share information on social networking sites.

When "replicability" is more than just "reliability": The Hubble constant controversy.

We propose that the epistemic functions of replication in science are best understood by relating them to kinds of experimental error/uncertainty. One kind of replication, which we call "direct replications," principally serves to assess the reliability of an experiment through its precision: the presence and degree of random error/statistical uncertainty. The other kind of replication, which we call "conceptual replications," principally serves to assess the validity of an experiment through its accuracy: the presence and degree of systematic errors/uncertainties. To illustrate the aptness of this general view, we examine the Hubble constant controversy in astronomy, showing how astronomers have responded to the concordances and discordances in their results by carrying out the different kinds of replication that we identify, with the aim of establishing a precise, accurate value for the Hubble constant. We contrast our view with Machery's "re-sampling" account of replication, which maintains that replications only assess reliability.

The inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in immunocompromised AG129 mice.

Zika virus, a mosquito-borne arbovirus, has repeatedly caused large pandemics with symptoms worsening from mild and self-limiting diseases to Guillain-Barré syndrome in adults and fetal microcephaly in newborns. In recent years, Zika virus diseases have posed a serious threat to human health. The shortage of susceptible small animal models makes it difficult to study pathogenic mechanisms and evaluate potential therapies for Zika virus infection. Therefore, we chose immunocompromised mice (AG129 mice) deficient in IFN-α/ß and IFN-γ receptors, which can abolish the innate immune system that prevents Zika virus infection early. AG129 mice were infected with the Zika virus, and this mouse model exhibited replication dynamics, tissue tropism, pathological lesion and immune activation of the Zika virus. Our results suggest that the inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in AG129 mice. By testing the potential antiviral drug favipiravir, several critical indicators, including replication dynamics and survival rates, were identified in AG129 mice after Zika virus infection. It is suggested that the model is reliable for drug evaluation. In brief, this model provides a potential platform for studies of the infectivity, virulence, and pathogenesis of the Zika virus. Moreover, the development of an accessible mouse model of Zika virus infection will expedite the research and deployment of therapeutics and vaccines.

Good Practice Guideline for Preclinical Alcohol Research: The STRINGENCY Framework.

Research in the field of preclinical alcohol research, but also science in general, has a problem: Many published scientific results cannot be repeated. As a result, findings from preclinical research often do not translate well to humans, causing increasing disappointment and calls for restructuring of preclinical research, that is, better reproducibility of preclinical research. However, the replication crisis is an inherent problem in biomedical research. Replication failures are not only due to small experimental variations but are often the result of poor methodology. In response to the replication crisis, numerous guidelines and recommendations have been proposed to promote transparency, rigor, and reproducibility in scientific research. What is missing today is a framework that integrates all the confusing information that results from all these guidelines and recommendations. Here we present STRINGENCY, an integrative approach to good practice guidelines for preclinical alcohol research, which can also apply to behavioral research in general and which aims to improve preclinical research to better prepare it for translation and minimize the "valley of death" in translational research. STRINGENCY includes systematic review and, when possible, meta-analysis prior to study design, sample size calculation, preregistration, multisite experiments, scientific data management (FAIR), reporting of data using ARRIVE, generalization of research data, and transparent publications that allow reporting of null results. We invite the scientific community to adopt STRINGENCY to improve the reliability and impact of preclinical alcohol research.

The role of gastropods in African swine fever virus ecology.

The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.

A glimpse into the hidden world of the flexible C-terminal protein binding domains of human RAD52.

Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.