Initiator Protein Research Articles

Mechanistic Origin of Cell-Size Control and Homeostasis in Bacteria

Evolutionarily divergent bacteria share a common phenomenological strategy for cell-size homeostasis under steady-state conditions. In the presence of inherent physiological stochasticity, cells following this "adder" principle gradually return to their steady-state size by adding a constant volume between birth and division, regardless of their size at birth. However, the mechanism of the adder has been unknown despite intense efforts. In this work, we show that the adder is a direct consequence of two general processes in biology: (1) threshold-accumulation of initiators and precursors required for cell division to a respective fixed number-and (2) balanced biosynthesis-maintenance of their production proportional to volume growth. This mechanism is naturally robust to static growth inhibition but also allows us to "reprogram" cell-size homeostasis in a quantitatively predictive manner in both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. By generating dynamic oscillations in the concentration of the division protein FtsZ, we were able to oscillate cell size at division and systematically break the adder. In contrast, periodic induction of replication initiator protein DnaA caused oscillations in cell size at initiation but did not alter division size or the adder. Finally, we were able to restore the adder phenotype in slow-growing E.coli, the only known steady-state growth condition wherein E.coli significantly deviates from the adder, by repressing active degradation of division proteins. Together, these results show that cell division and replication initiation are independently controlled at the gene-expression level and that division processes exclusively drive cell-size homeostasis in bacteria. VIDEO ABSTRACT.

Expansion and Evolutionary Patterns of Glycosyltransferase Family 8 in Gramineae Crop Genomes and Their Expression under Salt and Cold Stresses in Oryza sativa ssp. japonica.

Plant cell walls play a fundamental role in several ways, providing structural support for cells, resistance against pathogens and facilitating the communication between cells. The glycosyltransferase family 8 (GT8) is involved in the formation of the plant cell wall. However, the evolutionary relationship and the functional differentiation of this important gene family remain obscure in Gramineae crop genomes. In the present investigation, we identified 269 GT8 genes in the seven Gramineae representative crop genomes, namely, 33 in Hordeum vulgare, 37 in Brachypodium distachyon, 40 in Oryza sativa ssp. japonica, 41 in Oryza rufipogon, 36 in Setaria italica, 37 in Sorghum bicolor, and 45 in Zea mays. Phylogenetic analysis suggested that all identified GT8 proteins belonged to seven subfamilies: galacturonosyltransferase (GAUT), galacturonosyltransferase-like (GATL), GATL-related (GATR), galactinol synthase (GolS), and plant glycogenin-like starch initiation proteins A (PGSIP-A), PGSIP-B, and PGSIP-C. We estimated that the GAUT subfamily might be further divided into four subgroups (I–IV) due to differentiation of gene structures and expression patterns. Our orthogroup analysis identified 22 orthogroups with different sizes. Of these orthogroups, several orthogroups were lost in some species, such as S. italica and Z. mays. Moreover, lots of duplicate pairs and collinear pairs were discovered among these species. These results indicated that multiple duplication modes led to the expansion of this important gene family and unequal loss of orthogroups and subfamilies might have happened during the evolutionary process. RNA-seq, microarray analysis, and qRT-PCR analyses indicated that GT8 genes are critical for plant growth and development, and for stresses responses. We found that OsGolS1 was significantly up-regulated under salt stress, while OsGAUT21, OsGATL2, and OsGATL5 had remarkable up-regulation under cold stress. The current study highlighted the expansion and evolutionary patterns of the GT8 gene family in these seven Gramineae crop genomes and provided potential candidate genes for future salt- and cold- resistant molecular breeding studies in O. sativa.

A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein.

Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato yellow leaf curl virus (TYLCV), with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato golden mosaic virus (TGMV) to interact with the E2 SUMO-conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1 binding differs depending on the Rep protein examined.IMPORTANCE Nuclear activity of the replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization has been mapped for any geminiviral Rep protein. Our data add another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2-conjugating enzyme.

Structural analysis of a replication protein encoded by a plasmid isolated from a multiple sclerosis patient.

Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53 Å resolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α-helices, five β-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.

Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22.

Tailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed an in vitro P22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.IMPORTANCE The existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assembly in vivo indicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

Mobilisation of a small Acinetobacter plasmid carrying an oriT transfer origin by conjugative RepAci6 plasmids

Most Acinetobacter plasmids are genus specific but their properties have not been investigated. Small plasmids with Rep_3 family replication initiation proteins and iterons are common in Acinetobacter baumannii and often carry antibiotic resistance genes and toxin-antitoxin systems. A RepAci1 plasmid, carrying the carbapenem resistance gene oxa23 in Tn2006 and a RepAci2 plasmid carrying the amikacin (kanamycin and neomycin) resistance gene aphA6 in TnaphA6 were identified. These two plasmids have related rep regions; the consensus 22 bp iteron repeats differ only at three positions and the RepA proteins are 84% identical. However, they were shown to be compatible, whereas the RepAci1 plasmid displaced another RepAci1 plasmid demonstrating that they were incompatible. Despite encoding no mobilisation proteins, the RepAci1 plasmid was transferred to a new host at low frequency when a conjugatively proficient RepAci6 plasmid was present, whereas the RepAci2 plasmid carrying mobA and mobC mobilisation genes was not. Comparison of the sequences of the mobilised and mobilising plasmids revealed a short region of high similarity that is upstream of the predicted mobilisation genes in the RepAci6 plasmid, and has an organisation similar to that of F-type oriT transfer origins. The segment carrying the oriT-like region is present in many RepAci1 plasmids, including ones carrying the cabarpenem resistance genes oxa24 or oxa58 in dif modules, and in some RepAci2 or other Rep_3 plasmids of further types, including one carrying the tet39 tetracycline resistance determinant. These plasmids are also likely to be mobilised, spreading resistance.

Mutational Analysis of DnaA‐trio Motifs in E. coli OriC Reveals Multiple Modes for Bacterial Replication Origin Activation

The chromosomal replication origin (oriC) in bacteria is a unique region that directs assembly of the orisome, a nucleoprotein complex that unwinds origin DNA and assists with the loading of the replicative helicase. All orisome functions are mediated by the initiator protein, DnaA, which binds to specific sites in oriC, and, after DNA unwinding, binds to and stabilizes the unwound region. Orisome assembly is well characterized in E. coli, but bacterial origins are not conserved with regard to the number and spacing of DnaA recognition sites, and so it is difficult to determine whether there are common sequence patterns that are essential for origin activity. However, one promising feature, the “DnaA‐trio” motif, can be found in the origins of most bacterial types, with multiple copies residing near the AT‐rich region where DNA strand separation takes place. DnaA‐trios are essential for B. subtilis chromosome replication initiation, where they facilitate delivery of DnaA‐ATP oligomeric filaments to the AT‐rich region. Three DnaA‐trio motifs also reside in E. coli oriC, but their role is unclear, since we recently reported that E. coli oriC could be activated without formation of DnaA‐ATP oligomers. Therefore, to test their essentiality in E. coli, we used site‐directed mutagenesis to inactivate the trios, and then used recombineering to replace wild type chromosomal oriC with the mutated versions. Eliminating trio sites had no effect on E. coli oriC activity or initiation timing. Based on these results, we hypothesized that E. coli might use an alternative mechanism to deliver DnaA to the AT‐rich region, one that does not depend on trios or DnaA‐ATP filaments. We hypothesized that the sharp DNA bend formed late in E. coli orisome assembly and stabilized by the DNA bending protein IHF, might eliminate the need for trio sites by positioning the AT‐rich region for cross‐strand DnaA donations provided by the DnaA bound to oriC's left half. To test this, we removed the bend region from oriC using site‐directed mutagenesis, and even though the mutated origin (oriCno bend) contained all three DnaA trios, it was unable to function as the sole chromosomal origin of replication. In vitro unwinding studies suggested that the defect could be caused by a failure to deliver DnaA to the AT‐rich region, so we tried to rescue oriCno bend function by activating a trio‐dependent mode of DnaA delivery. Successful rescue was achieved by inserting a DnaA binding site between R1 and the trio sites, and origin activity became completely dependent on the DnaA‐trio elements. However, rescued cells displayed perturbed initiation timing, suggesting that the bend mode of DnaA delivery plays both mechanical and regulatory roles in E. coli. We propose that there are at least two possible modes of bacterial origin activation; one that depends on DnaA‐trio elements, and one that uses DNA bending and cross‐strand DnaA binding to regulate late stage orisome assembly.Support or Funding InformationNational Institutes of Health (GM54042 to A.L.) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to J.G.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acinetobacter and Moraxella OriCs are Functional as Chromosomal Replication Origin Transplants in E. coli

Bacterial chromosome replication is triggered by the initiator protein, DnaA, which assembles into a multimeric complex (orisome) that unwinds the replication origin, oriC, and helps to load the replicative DNA helicase. DnaA is highly conserved and the oriCs from nearly all bacteria carry clusters of oriC‐encoded DnaA recognition sites that guide step‐wise and properly timed orisome assembly. However, the arrangement of these sites is widely divergent among bacterial types and prior studies show that heterologous oriCs are only functional in extremely close relatives with nearly identical origin nucleotide sequence. To investigate the basis for DnaA recognition site diversity and possibly identify shared steps in orisome assembly, we replaced E. coli's chromosomal oriC with heterologous donor oriCs from its distant Gammaproteobacterial relatives and measured replication activity in vivo. Despite carrying little sequence similarity to the host, oriCs from Acinetobacter baylyi (ADP1) and Moraxella catarrhalis (ATCC 25240) were active in E. coli and used alternative pathways to assemble E. coli DnaA into functional orisomes. Mapping studies revealed the transplanted oriCs lacked the distinctive DNA unwinding element comprising 13mer repeat motifs as well as the arrayed low affinity DnaA recognition sites that are both hallmarks of E. coli oriC. Using flow cytometry analysis, we found that ADP1 and M. catarrhalis oriCs were also unable to initiate DNA synthesis at the proper cell cycle time in their E. coli hosts. Thus, the mechanisms for initiation timing must be distinct from those that permit replication fork assembly on these origins. We propose that E. coli and ADP1‐type orisomes share a sub‐architecture required for mechanical function (unwinding and helicase loading), but also carry type‐specific variations in the arrangement and affinity of DnaA recognition sites that are required for proper initiation timing using their cognate DnaAs. We propose that replication origin diversity reflects the many varieties of cell cycle‐coupled regulatory mechanisms used to control the availability/accessibility of DnaA at the oriCs of different bacterial types. Further studies of DnaA‐oriC interactions on heterologous origins may also reveal novel targets for broad spectrum bacterial growth inhibitors as well as those for type‐specific inhibition.Support or Funding InformationNational Institutes of Health (GM54042 to AL) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein Profile of Tissue Culture of TRI2025 Tea Clone

Tea is well known as favourite healthy drink for almost all people over the world. Tea propagation using conventional and modern ways are now developing rapidly. However, information regarding the protein profile of tissue culture of tea plant has not been revealed yet. This study aimed to determine the difference of protein profile of tea’s tissue culture using SDS-PAGE. This study was conducted using embryonic axes of TRI2025 tea clone cultured on MS media supplemented with 2,4-D for inducing somatic embryogenesis and globular-like structure (GLS) regeneration, and MS media supplemented with BAP for inducing shoot via organogenesis. The results revealed that proteins in the size of 37.69; 54.89; 60.77; 71.35; 87.34; and 92.99 KDa might be involved at somatic embryogenesis, and about 38.69 KDa, 69.27 KDa, and 55.76 KDa respectively for GLS, initiation of shoot, and initiation of GLS derived leaf. Predicted key protein for leaf initiation both directly or through GLS was about 31-33 KDa, while for callusing were about 27.56 and 52.73 KDa, and constitutive protein was about 22.75 KDa. This study provides the first report of protein profile of tea’s tissue culture. The information obtained can be beneficial as a marker for explant for somatic embryogenesis, GLS, or organogenesis pathway and as a scientific information for further biotechnology development.

Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway.

Ovarian cancer (OVCA) patients often develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase ‘a2' isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible factor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. The apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. These cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2-cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy flux. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V-ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer.

Profiling RNA‐Associated Proteins for Vasopressin‐Mediated Translation in Kidney Collecting Duct Cells

Vasopressin‐mediated gene expression changes in the renal collecting duct has been intensively studied over the last decade. Prior studies were focused on transcriptional regulation as a mechanism of regulation of transport by vasopressin. However, post‐transcriptional regulation at the RNA level has not been studied. To identify vasopressin‐mediated RNA modulators, 1) a comprehensive list of RNA‐associated proteins was curated, and 2) vasopressin‐regulated genes from this list were identified using RNA‐seq in mouse mpkCCD cells exposed to vasopressin for various times.The curated database of RNA‐associated proteins was generated by mining information from multiple databases as follows. RNA processing‐related proteins were identified from Gene Ontology (GO) ‘Molecular Function’ and ‘Biological Process’, KEGG pathway, RNA modification database and Reactome. In addition, proteins that have RNA‐binding domains were identified from five different databases (RBPDB, ATtRACT, RBPmap, InterPro and UniProt). A total of 1369 proteins are listed in the current database of RNA‐associated proteins (https://hpcwebapps.cit.nih.gov/ESBL/Database/RNAprocessing_Proteins/). Among these proteins, 646 proteins have known RNA‐binding domain structures and 723 proteins were identified with annotated molecular functions related to RNA processing. 371 proteins were identified by both criteria.RNA‐seq was carried out in mpkCCD cells treated with the vasopressin analog dDAVP (10−10 M) for 3, 6 and 12 h. Relative transcript abundance was estimated using transcripts per million (TPM) and change of transcript abundance was presented as Log2 of the dDAVP:vehicle TPM ratio at each time point. Interestingly, we found that transcript abundance of genes coding for ribosomal proteins (60S ribosomal proteins and 40S ribosomal proteins) and translation initiation proteins (eukaryotic translation initiation factors) were extensively changed by dDAVP at all time points. These results indicate that vasopressin‐mediated gene expression includes changes mediated by abundance changes of translation‐associated proteins. Beyond this, the current study provides new resources for further study of RNA‐associated processes in the kidney.Support or Funding InformationThe work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute projects ZIA‐HL001285 (to MAK) and ZIA‐HL006129 (to MAK).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prdm9 and Meiotic Cohesin Proteins Cooperatively Promote DNA Double-Strand Break Formation in Mammalian Spermatocytes

Meiotic recombination is required for correct segregation of chromosomes to gametes and to generate genetic diversity. In mice and humans, DNA double-strand breaks (DSBs) are initiated by SPO11 at recombination hotspots activated by PRDM9-catalyzed histone modifications on open chromatin. However, the DSB-initiating and repair proteins are associated with a linear proteinaceous scaffold called the chromosome axis, the core of which is composed of cohesin proteins. STAG3 is a stromalin subunit common to all meiosis-specific cohesin complexes. Mutations of meiotic cohesin proteins, especially STAG3, perturb both axis formation and recombination in the mouse, prompting determination of how the processes are mechanistically related. Protein interaction and genetic analyses revealed that PRDM9 interacts with STAG3 and REC8 in cooperative relationships that promote normal levels of meiotic DSBs at recombination hotspots in spermatocytes. The efficacy of the Prdm9-Stag3 genetic interaction in promoting DSB formation depends on PRDM9-mediated histone methyltransferase activity. Moreover, STAG3 deficiency has a major effect on DSB number even in the absence of PRDM9, showing that its role is not restricted to canonical PRDM9-activated hotspots. STAG3 and REC8 promote axis localization of the DSB-promoting proteins HORMAD1, IHO1, and MEI4, as well as SPO11 activity. These results establish that PRDM9 and axis-associated cohesin complexes together coordinate and facilitate meiotic recombination by recruiting key proteins for initiation of DSBs, thereby associating activated hotspots with DSB-initiating complexes on the axis.

Recent advances in understanding the replication initiator protein of the ssDNA plant viruses of the family Nanoviridae.

The families of viruses possessing single-stranded (ss) circular genome employ a dedicated replication initiator protein (Rep) for making copies of their genome through the process of rolling circle replication. The replication begins at conserved nonanucleotide sequence at the intergenic region. The Rep protein seems to be the most conserved amongst the available proteins of the nanovirids and comprises of the N-terminal endonuclease domain and the C-terminal helicase domain. The structural studies of Faba bean necrotic yellows virus endonuclease domain suggests a α + β fold comprising of central β sheet built from five antiparallel β strands surrounded by outer short α helices. The catalysis is mediated by a conserved Tyr residue and employs divalent metal ions (Mn2+). On one hand, the Reps associate with each other and oligomerize and on the other hand interact with varied host and vector associated proteins for successful infection. The sequence analysis of Reps from previously known nanovirids and the newly found ones from metagenomics data shed light on the evolutionary pattern of nanovirids in comparison to other plant infecting ssDNA viruses.

Iterons Homologous to Helper Geminiviruses Are Essential for Efficient Replication of Betasatellites.

Betasatellites associated with geminiviruses can be replicated promiscuously by distinct geminiviruses but exhibit a preference for cognate helper viruses. However, the cis elements responsible for betasatellite origin recognition have not been characterized. In this study, we identified an iteron-like repeated sequence motif, 5'-GAGGACC-3', in a tobacco curly shoot betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV). Competitive DNA binding assays revealed that two core repeats (5'-GGACC-3') are required for specific binding to TbCSV Rep; TbCSB iteron mutants accumulated to greatly reduced levels and lost the cognate helper-mediated replication preference. Interestingly, TbCSV also contains identical repeated sequences that are essential for specific Rep binding and in vivo replication. In order to gain insight into the mechanism by which TbCSB has acquired the cognate iterons, we performed a SELEX (systematic evolution of ligands by exponential enrichment) assay to identify the high-affinity Rep binding ligands from a large pool of randomized sequences. Analysis of SELEX winners showed that all of the sequences contained at least one core iteron-like motif, suggesting that TbCSB has evolved to contain cognate iterons for high-affinity Rep binding. Further analyses of various betasatellite sequences revealed a region upstream of the satellite conserved region replete with iterative sequence motifs, including species-specific repeats and a general repeat (5'-GGTAAAT-3'). Remarkably, the species-specific repeats in many betasatellites are homologous to those in their respective cognate helper begomoviruses, whereas the general repeat is widespread in most of the betasatellite molecules analyzed. These data, taken together, suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.IMPORTANCE The geminivirus-encoded replication initiator protein (Rep) binds to repeated sequence elements (also known as iterons) in the origin of replication that serve as essential cis elements for specific viral replication. Betasatellites associated with begomoviruses can be replicated by cognate or noncognate helper viruses, but the cis elements responsible for betasatellite origin recognition have not been characterized. Using a betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV) as a model, we identify two tandem repeats (iterons) in the Rep-binding motif (RBM) that are required for specific Rep binding and efficient replication, and we show that identical iteron sequences present in TbCSV are also necessary for Rep binding and the replication of helper viruses. Extensive analysis of begomovirus/betasatellite sequences shows that many betasatellites contain iteron-like elements homologous to those of their respective cognate helper begomoviruses. Our data suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.

Differential Roles of Pioneer Proteins in Initiation and Stabilization of Early Clathrin Coat Unveiled by a Novel Disassembly Bias Score

Differential Roles of Pioneer Proteins in Initiation and Stabilization of Early Clathrin Coat Unveiled by a Novel Disassembly Bias Score

Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein.

We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep). For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface. We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

Saliva as a biomarker for dental caries: A systematic review.

Aim:The aim of the present review article is to establish a systematic review to describe the role of various salivary components such as pH, buffering capacity, proteins, electrolyte, antioxidant, enzymes, and minerals in occurrence and initiation of dental caries in participants with and without dental caries.Methodology:An electronic search was conducted in the PubMed, Scopus, Web of Science, and Google Scholar databases. The MESH terms (Medical Subject Headings) were “dental caries susceptibility,” “caries risk assessment, ”Salivary Biomarker,” “saliva,” “Proteins,” “electrolytes,” “biomarker,” “Anti-oxidant’s.” The database search was limited to the studies published between 2008 and 2018 and only human studies were included. Furthermore, the STROBE criteria checklist was used to analyze the studies which had to be included in the systematic review.Results:A total of 150 articles were retrieved through electronic database. After evaluating the title, abstract, and full text of these articles, only 11 were selected for the present systematic review. However, two articles were excluded because they were classified as high risk of bias according to the STROBE criteria. Out of 9 studies selected, 5 were classified as low-risk bias and 4 were classified as moderate-risk bias.Conclusion:Based on the results of the systematic review, out of 11 studies, 7 found to have a statistically significant difference between individuals with and without caries experience. Hence, it can be concluded that there is an association between various components of saliva and dental caries.

DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction.

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA–DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum.

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40-50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.

HnRNP K: A Regulator of Global Transcription and Translation That Drives Lymphomagenesis

hnRNP K is an RNA binding protein that controls a multitude of cellular processes and is aberrantly expressed in cancers. We have previously shown that hnRNP K functions as a haploinsufficient tumor suppressor in AML patients with 9q deletions. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon. We have recently discovered that hnRNP K overexpression in patients with diffuse large B-cell lymphoma correlates with dismal outcomes and directly resulted in the development of lymphomas in transgenic mice.To understand the mechanistic basis for the oncogenicity of hnRNP K overexpression and to identify therapeutic vulnerabilities, we performed RNA-sequencing, RNA immunoprecipitation following by sequencing (RIP-Seq), mass spectrometry, and polysome assays. We observed that hnRNP K regulates both global transcription and translational processes within the cell via modulation of 7SK and translation initiation proteins (such as the eIFs and PABP), respectively. Consequently, we hypothesized that aberrant hnRNP K expression primarily perturbs oncogenes with short half-lives. Mechanistically, we identified that hnRNP K binds to the RNA and regulates the expression of a plethora of critical oncogenes and tumor suppressors involved in hematologic malignancies such as c-Myc, RUNX1, and Cyclin D1. As proof of concept for clinical applications, we have demonstrated that hnRNP K-driven c-Myc overexpression renders tumors susceptible to bromodomain inhibition.Given that hnRNP K directs global transcription and translation, it is likely that hnRNP K overexpressing tumors will also be sensitive to transcriptional and translational inhibitors such as CDK9 inhibitors and omacetaxine mepesuccinate, respectively. However, since hnRNP K also regulates a plethora of additional cellular processes that extend far beyond mRNA and protein synthesis, there is a need to develop hnRNP K-specific inhibitors that will only target these activities. Thus, we have recently begun to identify small molecule compounds that can directly inhibit hnRNP K-RNA binding function on specific targets using an in vitro fluorescent binding assay. Using this assay, we are currently screening a library of 70,000 small molecule compounds to identify agents that can prevent hnRNP K-RNA interactions.In summary, we have established that hnRNP K is a bona fide oncogene that drives lymphomagenesis. Global analyses have revealed therapeutic vulnerabilities of hnRNP K overexpressing tumors. Furthermore, using our in vitro RNA binding assays, we anticipate identification of novel hnRNP K-specific inhibitors. DisclosuresNo relevant conflicts of interest to declare.