T4 Gene Research Articles

BNO - An Ontology for Understanding the Transittability of Complex Biomolecular Networks

Analysis of biological systems is being progressively facilitated by computational tools. Most of these tools are based on qualitative and numerical methods. However, they are not always evident, and there is an increasing need to provide an additional semantic layer. Semantic technologies, especially ontologies, are one of the tools frequently used for this purpose. Indeed, they are indispensable for understanding the semantic knowledge about the operation of cells at a molecular level. We describe here the biomolecular network ontology (BNO) created specially to address the needs of analysing the complex biomolecular network's behaviour. A biomolecular network consists of nodes, denoting cellular entities, and edges, representing interactions among cellular components. The BNO ontology provides a foundation for qualitative simulation of complex biomolecular networks. We test the performance of the proposed BNO ontology by using a real example of a biomolecular network, the bacteriophage T4 gene 32. We illustrate the proposed BNO ontology for reasoning and inferring new knowledge with sets of rules expressed in SWRL. Results demonstrate that the BNO ontology allows to precisely interpret the corresponding semantic context and intelligently model biomolecular networks and their state changes. The Biomolecular Network Ontology (BNO) is freely available at https://github.com/AliAyadi/BNO-ontology-version-1.0.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.

The Interaction between Bacteriophage T7 DNA Polymerase and Gene 2.5 Protein at the Single-Molecule Level

The Interaction between Bacteriophage T7 DNA Polymerase and Gene 2.5 Protein at the Single-Molecule Level

Integrated transcriptomic analysis of Trichosporon Asahii uncovers the core genes and pathways of fluconazole resistance

Trichosporon asahii (T. asahii) has emerged as a dangerous pathogen that causes rare but life-threatening infections. Its resistance to certain antifungal agents makes it difficult to treat, especially for patients undergoing long-term antibiotic therapy. In this study, we performed a series of fluconazole (FLC) perturbation experiments for two T. asahii strains, a clinical isolate stain CBS 2479 (T2) and an environmental isolate strain CBS 8904 (T8), to uncover potential genes and pathways involved in FLC resistance. We achieved 10 transcriptomes of T2 and T8 that were based on dose and time series of FLC perturbations. Systematic comparisons of the transcriptomes revealed 32 T2 genes and 25 T8 genes that are highly sensitive to different FLC perturbations. In both T2 and T8 strains with the phenotype of FLC resistance, the processes of oxidation-reduction and transmembrane transport were detected to be significantly changed. The antifungal susceptibility testing of FLC and penicillin revealed their resistance pathways are merged. Accumulated mutations were found in 564 T2 and 225 T8 genes, including four highly mutated genes that are functionally related to the target of rapamycin complex (TOR). Our study provides abundant data towards genome-wide understanding of the molecular basis of FLC resistance in T. asahii.

Gene Expression Correlated with Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease.

Severe asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals. Identify networks of genes reflective of underlying biological processes that define SA. Airway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders. Weighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12-21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA. In this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes.

BNO: An ontology for describing the behaviour of complex biomolecular networks

The use of semantic technologies, such as ontologies, to describe and analyse biological systems is at the heart of systems biology. Indeed, understanding the behaviour of cells requires a large amount of context information. In this paper, we propose an ontology entitled ”Biomolecular Network ontology” using the OWL language. The BNO ontology standardises the terminology used by biologists experts to address issues including semantic behaviour representation, reasoning and knowledge sharing. The main benefit of this proposed ontology is the ability to reason about dynamical behaviour of complex biomolecular networks over time. We demonstrate our proposed ontology with a detailed example, the bacteriophage T4 gene 32 use case.

CBNSimulator: a simulator tool for understanding the behaviour of complex biomolecular networks using discrete time simulation

Because of the lack of satisfactory solutions to explain biological systems, biologists usually focus on modelling and simulation tools to understand the behaviour of these complex organisms. Indeed, computational modelling and simulation of cells plays a pivotal role in systems biology. In this paper, we tackle the problem of studying the behaviour of human cells by reproducing the behaviour of complex biomolecular networks. To this end, we present in this paper an approach for simulating complex biomolecular networks inspired by the discrete-event simulation model (DEVS), a formalism developed for supporting the modelling of complex systems. In this paper, we propose a simulation tool, named ”CBNSimulator”, based on a logical model of the biomolecular network and taking advantage of the performance of a discrete-time simulation model for understanding the evolution and the behaviour of complex biomolecular networks as a discrete sequence of events in time. The proposed tool has been applied to the case study of a ribosomal protein regulation network, named ”the bacteriophage T4 gene 32”, and results given by this simulation tool are in agreement with the expert’s judgement. Moreover, the graphical user interface of CBNSimulator allows biologists to easily reproduce, analyse and understand behaviour of complex biomolecular networks through discrete simulation.

Bacteriophage T5 gene D10 encodes a branch-migration protein.

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.

Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective.

Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. “Hibernation” mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a “scavenger” response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems able to produce no more than an average of one phage per originally available cell, and few if any further progeny are produced by cells in this mode even if fresh nutrients are made available later.

Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference.

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.

Evaluation of possible occurrence of mutation in MMR repair system genes in resistant and sensitiveclinical strains of Mycobacterium tuberculosisby using sequencing method

Evaluation of possible occurrence of mutation in MMR repair system genes in resistant and sensitiveclinical strains of Mycobacterium tuberculosisby using sequencing method AmirPoyan Afzali1, Mohammad Arjomndzadegan2, Azam Ahmadi 3, Sayead Hossein Hosseini1, Manijeh Kahbazi4 , Mojtaba Tousheh5 1- Department of Microbiology, Faculty of Science, Islamic Azad University of Tehran Research 2- Infectious Diseases Research center (IDRC) and Department of Microbiology and Immunology, School of medicine, Arak University of Medical Sciences, Arak, Iran 3- Department of molecular genetics, Tarbiat Modarres University, Tehran and Infectious Diseases Research center (IDRC), Arak University of Medical Sciences, Arak , Iran , azam.ahmadi@modares.ac.ir 4- Infectious Diseases Research center (IDRC), Department of Pediatric Infectious, Arak University of Medical Sciences, Arak, Iran 5- Department of Cellular and Molecular Biology, Faculty of Science, University of Isfahan Abstract: Background:during recent years, the incidence and spread of drug resistance in Mycobacterium tuberculosis, the bacterium causing tuberculosis, has set this disease in World Health Organizationpriorities alignment of diseases like AIDS and hepatitis. Study of close examination of resistant and susceptible clinical strains genotypes is necessary to overcome drug resistance. Among the numerous repair systems, only there are limited number of encoding genes of DNA repair enzymes in Mycobacterium tuberculosis. Commonly these genes have been conserved and any changes among them likely increasethe mutation occurance due to the impossibility of correctionof spontaneous mutations insensitive strains of this bacteria.mut genes encodeDNA repairable enzymes.This study investigated the mutations in these genes and the effect of these mutations on tuberculosis drug resistance. Materials&Methods: In this study,of 29 available specimens,we were selected 8 susceptible strains and 21 resistantstrains andafter ordering appropriate primers and performing the proliferation reaction two types of amplicons produced which includingfragments of genes mut T2 and mut T4 and they were sent inorder to sequencing. Results:The results of chain reactionprimer represents an appropriate choice of primerswhich were investigated. Sequencing results showed that overall 73% of resistant strains that had been selected for study of mutT4gene, have no mutations in codons 48of mutT4 gene, and 70% of resistant strains have no GGA >>> CGA mutation at codon 58 of mutT2 gene. Conclusion: One of the strategies to overcome tuberculosis drug resistance is a close examination of genotypes of resistant and susceptible clinical strains. Results of this study was performedby examining changes in mut T2 and mut T4 gene sequence. The mutation in mut T2 always associated with mutation in mut T4, in this way, the first mutation may occurs in mut T4and after that, the second mutationmay occurs in mut T2. Importance of this is determined by study of encoded proteins by these two genes and position of mut t4thanmut T2 in the MUT HLS spatial structure of protein complex. Results of comparison of drug resistance and occurrence of mutations in hot-spots of mut T2 and mut T4 genes illustrated that these genes are conserved in resistant strains. However, there is no significant relation in susceptible strains. Keywords: Mycobacterium tuberculosis, mut T2, PCR, sequencing

Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus.

Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.

Chloroplast mRNA translation is regulated by the 5'-untranslated region (5'-UTR). Chloroplast 5'-UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5'-UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5'-UTR with the E. coli phage T7 gene 10 5'-UTR, a highly active 5'-UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5'-UTR with a cognate 5'-coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5'-UTR and its coding region is important for translational initiation.

Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions

Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3' of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements.

DNA Binding Proteins and Drug Delivery Vehicles: Tales of Elephants and Snakes.

We compare the DNA-interactive properties of bacteriophage T4 gene 32 protein (gp32) with those of crotamine, a component of the venom of the South American rattlesnake. Gene 32 protein is a classical single-stranded DNA binding protein that has served as a model for this class of proteins. We discuss its biological functions, structure, binding specificities, and how it controls its own expression. In addition, we delineate the roles of the structural domains of gp32 and how they regulate the protein's various activities. Crotamine, a component of the venom of the South American rattlesnake, is probably not a DNA binding protein in nature, but clearly shows significant DNA binding in vitro. Crotamine has been shown to selectively disrupt rapidly dividing cells and this specificity has been demonstrated for crotamine-facilitated delivery of plasmid DNA Thus, crotamine, or a variant of the protein, could have important clinical and/or diagnostic roles. Understanding its DNA binding properties may therefore lead to more effective drug delivery vehicles.

T4 Factor Involved in Phage‐induced Host mRNA Degradation

ObjectiveIdentification and characterization of T4 factor involved in phage‐induced host mRNA degradationResultsBacteriophage T4 causes a shift of gene expression from host Escherichia coli to T4 immediately after infection. In this process, host mRNAs are rapidly degraded, which may facilitate T4 gene expression by providing precursors for transcription and free ribosomes. Recently we found that the rapid degradation of host mRNAs was partially suppressed and a decay intermediate of lpp mRNA was markedly accumulated in Δ(39‐56)6 phage‐infected cells. Since the 4.6‐kb region missing in Δ(39‐56)6 phage contain 8 orfs, we tested the effect of each orf on host mRNA degradation and found that the causal gene is dda.2, a T4 early gene, located immediate downstream of an early promoter. 
 The average number of phages produced in a cell infected with a deletion mutant of dda.2 (Δdda.2) was one‐third, in comparison to that of wild type, indicating that dda.2 is necessary for efficient propagation of T4. On the other hand, over‐expression of Dda.2 was found to be toxic for cells. With the induction of plasmid‐borne dda.2, cell growth was stopped within 30 min, and cell viability was significantly reduced. In addition, amounts of E. coli mRNAs, such as lpp and ompA were considerably reduced. Furthermore, the decay of ompA and lpp mRNAs was accelerated after induction of plasmid‐borne dda.2.Conclusions1. T4 dda.2 is involved in phage‐induced host mRNA degradation, which is thought to be important to facilitate T4 gene expression.2. T4 dda.2 has a significant role in bacterial physiology probably with a modulation in mRNA metabolism.Source of the research support: The ministry of education, culture, sports, science and technology, Japan

Unraveling the Interplay between Single-Stranded DNA-Binding Protein, DNA Polymerase and Single-Stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5), a single-stranded DNA binding (SSB) protein, contains two main structural elements which confer it essential functions. First, an oligosaccharide/oligonucleotide binding fold which can interact with ssDNA and thus provide a protective role while removing secondary structure impediments. Second, an acidic C-terminal tail capable to interact with other gp2.5 units and with other replisome proteins, such as the T7 DNA primase-helicase (gp4) and polymerase (gp5), allowing for organizational roles within the replisome. The impact of SSBs on the replisome dynamics is hard to study using conventional biochemistry tools. Here, we present data obtained from a unique combination of optical tweezers and confocal fluorescence microscopy, which offers real-time high spatial and temporal resolution to study protein-DNA interactions. Our results show that the SSB gp2.5 binds efficiently to ssDNA, forming highly static protein-DNA complexes. We also observed a decrease in the ssDNA end-to-end length indicating that the protein binds by bending or wrapping ssDNA, resulting in a highly tension-sensitive binding mode. Next, we studied to what degree the presence of wild type (wt) gp2.5 or a mutant lacking the terminal phenylalanine (ΔF), the interaction motif with DNA polymerase, affects replication activity. We show that wt gp2.5 alters the rate, duration and probability of occurrence of replicative and proofreading polymerase events in contrast to the ΔF mutant or the absence of the protein. Thus, our findings indicate that the presence of gp2.5 strongly stimulates the function of DNA polymerase within the replisome.

Repetitive Single-Molecule FRET Fluctuations upon T4 Gene 32 Protein Binding to Single-Stranded DNA

Bacteriophage T4-coded gene 32 protein (gp32) is an essential component of the T4 DNA replication, recombination, and repair systems. Gp32 is a single-stranded DNA binding protein that binds cooperatively to single-stranded (ss) DNA templates exposed by the function of the T4 primosome helicase, and configures these templates for efficient use by the replisome. To this end gp32 binding helps melt out transiently formed ssDNA secondary structures, protects exposed ssDNA sequences from nucleases, and helps to integrate the functions of the other components of the replication complex. It has been well studied by bulk biochemical methods for about 40 years. However, many aspects of the detailed mechanisms -- and especially the dynamics -- of the interactions of gp32 with both double-stranded (ds) and single-stranded DNA are still not well studied or well understood.We have used single-molecule Forster Resonance Energy Transfer (sm-FRET) methods to monitor the cooperative and non-cooperative binding of gp32 to the ssDNA sequences of model DNA replication forks, and have shown that the observed binding dynamics depend significantly on gp32 concentration, salt concentration and ssDNA segment length. Upon addition of 1 µM concentrations of gp32 into a sample chamber containing tethered single molecule replication fork constructs, we observed the appearance of ‘repetitive FRET fluctuations’ of the individual DNA molecules (>70% of the molecules per imaging area) on a 100 ms timescale. We noted also that these repetitive FRET fluctuations were substantially less prominent under the conditions of tight and fully cooperative gp32 protein binding. We have used ensemble and single-molecule fluorescence approaches to probe the dynamics of these gp32-ssDNA interactions in detail, and will discuss possible molecular interpretations of these smFRET fluctuations in terms of potential reaction intermediates and association-dissociation pathways in real time.

Phylogenetic analyses and characterization of RNase X25 from Drosophila melanogaster suggest a conserved housekeeping role and additional functions for RNase T2 enzymes in protostomes.

Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in Drosophila melanogaster. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by in gel assays, and has an acidic pH preference. Gene expression analyses showed that the RNase X25 transcript is present in all adult tissues and developmental stages. RNase X25 expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of RNase X25 expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the D. melanogaster genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.