Strand-switching mechanism of Pif1 helicase induced by its collision with a G-quadruplex embedded in dsDNA

G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.

BI-2536 Promotes Neuroblastoma Cell Death via Minichromosome Maintenance Complex Components 2 and 10

DNA replication is initiated with the recognition of the starting point of multiple replication forks by the origin recognition complex and activation of the minichromosome maintenance complex 10 (MCM10). Subsequently, DNA helicase, consisting of the MCM protein subunits MCM2-7, unwinds double-stranded DNA and DNA synthesis begins. In previous studies, replication factors have been used as clinical targets in cancer therapy. The results showed that MCM2 could be a proliferation marker for numerous types of malignant cancer. We analyzed samples obtained from patients with neuroblastoma, revealing that higher levels of MCM2 and MCM10 mRNA were associated with poor survival rate. Furthermore, we combined the results of the perturbation-induced reversal effects on the expression levels of MCM2 and MCM10 and the sensitivity correlation between perturbations and MCM2 and MCM10 from the Cancer Therapeutics Response Portal database. Small molecule BI-2536, a polo-like kinase 1 (PLK-1) inhibitor, is a candidate for the inhibition of MCM2 and MCM10 expression. To test this hypothesis, we treated neuroblastoma cells with BI-2536. The results showed that the drug decreased cell viability and reduced the expression levels of MCM2 and MCM10. Functional analysis further revealed enrichments of gene sets involved in mitochondria, cell cycle, and DNA replication for BI-2536-perturbed transcriptome. We used cellular assays to demonstrate that BI-2536 promoted mitochondria fusion, G2/M arrest, and apoptosis. In summary, our findings provide a new strategy for neuroblastoma therapy with BI-2536.

Clinical, Histological, and Genetic Features of 25 Patients with Autosomal Dominant Progressive External Ophthalmoplegia (ad-PEO)/PEO-Plus Due to TWNK Mutations

Autosomal dominant mutations in the TWNK gene, which encodes a mitochondrial DNA helicase, cause adult-onset progressive external ophthalmoplegia (PEO) and PEO-plus presentations. In this retrospective observational study, we describe clinical and complementary data from 25 PEO patients with mutations in TWNK recruited from the Hospital 12 de Octubre Mitochondrial Disorders Laboratory Database. The mean ages of onset and diagnosis were 43 and 63 years, respectively. Family history was positive in 22 patients. Ptosis and PEO (92% and 80%) were the most common findings. Weakness was present in 48%, affecting proximal limbs, neck, and bulbar muscles. Exercise intolerance was present in 28%. Less frequent manifestations were cardiac (24%) and respiratory (4%) involvement, neuropathy (8%), ataxia (4%), and parkinsonism (4%). Only 28% had mild hyperCKemia. All 19 available muscle biopsies showed signs of mitochondrial dysfunction. Ten different TWNK mutations were identified, with c.1361T>G (p.Val454Gly) and c.1070G>C (p.Arg357Pro) being the most common. Before definitive genetic confirmation, 56% of patients were misdiagnosed (36% with myasthenia, 20% with oculopharyngeal muscle dystrophy). Accurate differential diagnosis and early confirmation with appropriately chosen complementary studies allow genetic counseling and the avoidance of unnecessary treatments. Thus, mitochondrial myopathies must be considered in PEO/PEO-plus presentations, and particularly, TWNK is an important cause when positive family history is present.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Detecting the Different Responses of Roots and Shoots to Gravity in Salix matsudana (Koidz)

The study of the gravity response of roots and shoots is of great significance when exploring the polarity of plants and the development of the forest industry. In our study, normal and inverted cuts of Salix matsudana (Koidz) were cultured. The total RNAs of roots and shoots were extracted. Based on the comparative transcriptome, 412 and 668 genes were differentially expressed. The plasma membrane-, cell wall-, and extracellular region-related genes were up-regulated in the shoots, while the carbon metabolism and the nitrogen metabolism were up-regulated in the roots. Combining the alternative splicing genes, we found a potential gravity response network: in the shoots, LecRLKs were highly sensitive to gravity and further affected the alternative splicing of SNARE, as well as inducing an inhomogeneous distribution of auxin and a negative geotropism curve. In the roots, AP2/ERFs and STRKs were highly sensitive to gravity and regulated the expression level of STPKs and WAKs, finally resulting in a geotropism curve. Moreover, cell division was suppressed in both the roots and the shoots under inverted conditions with different mechanisms. Cell division inhibitors (KRPs) were up-regulated in the roots, while DNA helicase MCMs were down-regulated in the shoots. These results provide an important foundation for further studies of the molecular mechanisms and genetic regulation of plant responses to gravity and the plant polarity of forest trees.

Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection

Background The Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation. Results In this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells. Conclusions Our findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.

Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

Molecular Mechanisms of the RECQ4 Pathogenic Mutations

The human RECQ4 gene encodes an ATP-dependent DNA helicase that contains a conserved superfamily II helicase domain located at the center of the polypeptide. RECQ4 is one of the five RECQ homologs in human cells, and its helicase domain is flanked by the unique amino and carboxyl termini with sequences distinct from other members of the RECQ helicases. Since the identification of the RECQ4 gene in 1998, multiple RECQ4 mutations have been linked to the pathogenesis of three clinical diseases, which are Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO. Patients with these diseases show various developmental abnormalities. In addition, a subset of RECQ4 mutations are associated with high cancer risks, especially for osteosarcoma and/or lymphoma at early ages. The discovery of clinically relevant RECQ4 mutations leads to intriguing questions: how is the RECQ4 helicase responsible for preventing multiple clinical syndromes? What are the mechanisms by which the RECQ4 disease mutations cause tissue abnormalities and drive cancer formation? Furthermore, RECQ4 is highly overexpressed in many cancer types, raising the question whether RECQ4 acts not only as a tumor suppressor but also an oncogene that can be a potential new therapeutic target. Defining the molecular dysfunctions of different RECQ4 disease mutations is imperative to improving our understanding of the complexity of RECQ4 clinical phenotypes and the dynamic roles of RECQ4 in cancer development and prevention. We will review recent progress in examining the molecular and biochemical properties of the different domains of the RECQ4 protein. We will shed light on how the dynamic roles of RECQ4 in human cells may contribute to the complexity of RECQ4 clinical phenotypes.

Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage

Shigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.