Control of Synaptic Levels of Nicotinic Acetylcholine Receptor by the Sequestering Subunit Dα5 and Secreted Scaffold Protein Hig.

The presentation of nicotinic acetylcholine receptors (nAChRs) on synaptic membranes is crucial for generating cholinergic circuits, some of which are associated with memory function and neurodegenerative disorders. Although the physiology and structure of nAChR, a cation channel comprising five subunits, have been extensively studied, little is known about how the receptor levels in interneuronal synapses are determined and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices and intracellular proteins. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts in the brain. Only the loss of function of Dα5 among the 10 nAChR subunits suppressed hig mutant phenotypes in both male and female flies. Dα5 behaved as a lethal factor when Hig was defective; loss of Dα5 in hig mutants rescued lethality, upregulating Dα6 synaptic levels. By contrast, levels of Dα5, Dα6, and Dα7 subunits were all reduced in hig mutants. These three subunits have distinct properties for interaction with Hig or trafficking, as confirmed by chimeric subunit experiments. Notably, the chimeric Dα5 protein, which has the extracellular sequences that display no positive interaction with Hig, exhibited abnormal distribution and lethality even in the presence of Hig. We propose that the sequestering subunit Dα5 functions by reducing synaptic levels of nAChR through internalization, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix.SIGNIFICANCE STATEMENT Because the cholinergic synapse is one of the major synapses that generate various brain functions, numerous studies have sought to reveal the physiology and structure of the nicotinic acetylcholine receptor (nAChR). However, little is known about how synaptic levels of nAChR are controlled and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts. Our data indicate that Dα5 functions in reducing synaptic levels of nAChR, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix.

Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis.

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. Here, we reveal a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, our in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.

Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model.

APP (amyloid precursor protein), the causative molecule of Alzheimer's disease, is synthesized in neuronal cell bodies and subsequently transported to synapses. We previously showed that the yata gene is required for the synaptic transport of the APP orthologue in Drosophila melanogaster. In this study, we examined the effect of a reduction in yata expression in the Drosophila Alzheimer's disease model, in which expression of human mutant APP was induced. The synaptic localization of APP and other synaptic proteins was differentially inhibited by yata knockdown and null mutation. Expression of APP resulted in abnormal synaptic morphology and the premature death of animals. These phenotypes were partially but significantly rescued by yata knockdown, whereas yata knockdown itself caused no abnormality. Moreover, we observed that synaptic transmission accuracy was impaired in our model, and this phenotype was improved by yata knockdown. Thus, our data suggested that the phenotypes caused by APP can be partially prevented by inhibition of the synaptic localization of a subset of synaptic proteins including APP.

The Matrix Proteins Hasp and Hig Exhibit Segregated Distribution within Synaptic Clefts and Play Distinct Roles in Synaptogenesis.

The synaptic cleft is the space through which neurotransmitters convey neural information between two synaptic terminals. This space is presumably filled with extracellular matrix molecules involved in synaptic function or differentiation. However, little is known about the identities of the matrix components, and it remains unclear how these molecules organize the matrix in synaptic clefts. In this study, we identified Hasp, a Drosophila secretory protein containing CCP and WAP domains. Molecular genetic analysis revealed that Hasp diffuses extracellularly and is predominantly captured at synaptic clefts of cholinergic synapses. Furthermore, Hasp regulates levels of DLG and the nAChR subunits Dα6 and Dα7 at postsynaptic terminals. Hasp is required for trapping of another matrix protein, Hig, which is also secreted and diffused in the brain, at synaptic clefts of cholinergic synapses; however, Hig is dispensable for localization of Hasp at synaptic clefts. In addition, in the brains of triple mutants for the nAChR subunits Dα5, Dα6, and Dα7, the level of Hig, but not Hasp, was markedly reduced in synaptic regions, indicating that these nAChR subunits are required to anchor Hig to synaptic clefts. High-resolution microscopy revealed that Hasp and Hig exhibit segregated distribution within individual synaptic clefts, reflecting their differing roles in synaptogenesis. These data provide insight into how Hasp and Hig construct the synaptic cleft matrix and regulate the differentiation of cholinergic synapses, and also illuminate a previously unidentified architecture within synaptic clefts. SIGNIFICANCE STATEMENT: The synapse has been extensively studied because it is essential for neurotransmission. By contrast, the space between the synaptic terminals, the synaptic cleft, is still an undeveloped research area despite its ubiquity in synapses. In fruit fly brains, we obtained evidence that the matrix protein Hasp and the previously identified Hig, both of which are secreted extracellularly, localize predominantly to synaptic clefts of cholinergic synapses, and modulate the levels of nAChR subunits on postsynaptic membranes. However, Hasp and Hig play differential roles in matrix formation and exhibit segregated distribution within synaptic clefts. These results reveal the molecular mechanisms of synaptic matrix construction and illuminate a molecular architecture within synaptic clefts previously unrevealed in any animal species.

The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain.

The synaptic cleft, a crucial space involved in neurotransmission, is filled with extracellular matrix that serves as a scaffold for synaptic differentiation. However, little is known about the proteins present in the matrix and their functions in synaptogenesis, especially in the CNS. Here, we report that Hikaru genki (Hig), a secreted protein with an Ig motif and complement control protein domains, localizes specifically to the synaptic clefts of cholinergic synapses in the Drosophila CNS. The data indicate that this specific localization is achieved by capture of secreted Hig in synaptic clefts, even when it is ectopically expressed in glia. In the absence of Hig, the cytoskeletal scaffold protein DLG accumulated abnormally in cholinergic postsynapses, and the synaptic distribution of acetylcholine receptor (AchR) subunits Dα6 and Dα7 significantly decreased. hig mutant flies consistently exhibited resistance to the AchR agonist spinosad, which causes lethality by specifically activating the Dα6 subunit, suggesting that loss of Hig compromises the cholinergic synaptic activity mediated by Dα6. These results indicate that Hig is a specific component of the synaptic cleft matrix of cholinergic synapses and regulates their postsynaptic organization in the CNS.

A gain-of-function screen to identify genes that reduce lifespan in the adult of Drosophila melanogaster.

BACKGROUND: Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer's, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual's life, but can cause physiological defects and disease when misexpressed in adulthood. RESULTS: We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 "reduced-lifespan genes" that, when misexpressed in adulthood, shortened the flies' lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development. CONCLUSIONS: We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes.

Discovering irregular pupil light responses to chromatic stimuli using waveform shapes of pupillograms.

BACKGROUND: The waveforms of the pupillary light reflex (PLR) can be analyzed in a diagnostic test that allows for differentiation between disorders affecting photoreceptors and disorders affecting retinal ganglion cells, using various signal processing techniques. This procedure has been used on both healthy subjects and patients with age-related macular degeneration (AMD), as a simple diagnostic procedure is required for diagnosis. RESULTS: The Fourier descriptor technique is used to extract the features of PLR waveform shapes of pupillograms and their amplitudes. To detect those patients affected by AMD using the extracted features, multidimensional scaling (MDS) and clustering techniques were used to emphasize stimuli and subject differences. The detection performance of AMD using the features and the MDS technique shows only a qualitative tendency, however. To evaluate the detection performance quantitatively, a set of combined features was created to evaluate characteristics of the PLR waveform shapes in detail. Classification performance was compared across three categories (AMD patients, aged, and healthy subjects) using the Random Forest method, and weighted values were optimized using variations of the classification error rates. The results show that the error rates for healthy pupils and AMD-affected pupils were low when the value of the coefficient for a combination of PLR amplitudes and features of waveforms was optimized as 1.5. However, the error rates for patients with age-affected eyes was not low. CONCLUSIONS: A classification procedure for AMD patients has been developed using the features of PLR waveform shapes and their amplitudes. The results show that the error rates for healthy PLRs and AMD PLRs were low when the Random Forest method was used to produce the classification. The classification of pupils of patients with age-affected eyes should be carefully considered in order to produce optimum results.

A survey of scientific literacy to provide a foundation for designing science communication in Japan.

There are various definitions and survey methods for scientific literacy. Taking into consideration the contemporary significance of scientific literacy, we have defined it with an emphasis on its social aspects. To acquire the insights needed to design a form of science communication that will enhance the scientific literacy of each individual, we conducted a large-scale random survey within Japan of individuals older than 18 years, using a printed questionnaire. The data thus acquired were analyzed using factor analysis and cluster analysis to create a 3-factor/4-cluster model of people's interest and attitude toward science, technology and society and their resulting tendencies. Differences were found among the four clusters in terms of the three factors: scientific factor, social factor, and science-appreciating factor. We propose a plan for designing a form of science communication that is appropriate to this current status of scientific literacy in Japan.

RecQ5 interacts with Rad51 and is involved in resistance of Drosophila to cisplatin treatment.

RecQ5 is a member of the RecQ family of DNA helicases. There are 5 RecQ members in humans. Defects in 3 of them, i.e., BLM, WRN, and RTS, cause Bloom, Werner, and Rothmund-Thomson syndromes, respectively. RECQL1 and RECQL5 have not been associated with any human disease, and their precise roles are unknown. Our previous study suggests that the lack of RecQ5, which is the Drosophila homolog of RECQL5, leads to the accumulation of DNA double-stranded breaks (DSBs). It is possible that RecQ5 is involved in DSB repair. However, little is known about this possible function of RecQ5 in DSB repair. Here, we report that Rad51 protein, which plays a critical role in DSB repair, interacted with RecQ5 in vitro and in vivo in Drosophila. The Rad51 protein interacted with the C-terminal region of RecQ5, as shown by the yeast two-hybrid method. Moreover, the C-terminal region of the RecQ5 protein and the central region of Rad51 interacted directly and specifically when examined by the glutathione-S-transferase pull-down method. Consistent with these results, when RecQ5 and Rad51 were co-expressed in Drosophila cells in culture, they became co-localized in nuclei and could be co-immunoprecipitated. Furthermore, RecQ5-deficient flies (recq5) were more sensitive to the chemotherapeutic agent cisplatin compared with wild-type ones. Also, Rad51 mutants (rad51) were more sensitive to cisplatin, with sensitivity similar to that of recq5 rad51 double mutants. These data suggest that RecQ5 and Rad51 in Drosophila functioned for survival after the flies had been treated with cisplatin.

Drosophila carrying pex3 or pex16 mutations are models of Zellweger syndrome that reflect its symptoms associated with the absence of peroxisomes.

The peroxisome biogenesis disorders (PBDs) are currently difficult-to-treat multiple-organ dysfunction disorders that result from the defective biogenesis of peroxisomes. Genes encoding Peroxins, which are required for peroxisome biogenesis or functions, are known causative genes of PBDs. The human peroxin genes PEX3 or PEX16 are required for peroxisomal membrane protein targeting, and their mutations cause Zellweger syndrome, a class of PBDs. Lack of understanding about the pathogenesis of Zellweger syndrome has hindered the development of effective treatments. Here, we developed potential Drosophila models for Zellweger syndrome, in which the Drosophila pex3 or pex16 gene was disrupted. As found in Zellweger syndrome patients, peroxisomes were not observed in the homozygous Drosophila pex3 mutant, which was larval lethal. However, the pex16 homozygote lacking its maternal contribution was viable and still maintained a small number of peroxisome-like granules, even though PEX16 is essential for the biosynthesis of peroxisomes in humans. These results suggest that the requirements for pex3 and pex16 in peroxisome biosynthesis in Drosophila are different, and the role of PEX16 orthologs may have diverged between mammals and Drosophila. The phenotypes of our Zellweger syndrome model flies, such as larval lethality in pex3, and reduced size, shortened longevity, locomotion defects, and abnormal lipid metabolisms in pex16, were reminiscent of symptoms of this disorder, although the Drosophila pex16 mutant does not recapitulate the infant death of Zellweger syndrome. Furthermore, pex16 mutants showed male-specific sterility that resulted from the arrest of spermatocyte maturation. pex16 expressed in somatic cyst cells but not germline cells had an essential role in the maturation of male germline cells, suggesting that peroxisome-dependent signals in somatic cyst cells could contribute to the progression of male germ-cell maturation. These potential Drosophila models for Zellweger syndrome should contribute to our understanding of its pathology.

Modulation of neurotransmitter receptors and synaptic differentiation by proteins containing complement-related domains.

Neurotransmitter receptors play central roles in basic neurotransmission and synaptic plasticity. Recent studies have revealed that some transmembrane and extracellular proteins bind to neurotransmitter receptors, forming protein complexes that are required for proper synaptic localization or gating of core receptor molecules. Consequently, the components of these complexes contribute to long-term potentiation, a process that is critical for learning and memory. Here, we review factors that regulate neurotransmitter receptors, with a focus on proteins containing CUB (complement C1r/C1s, Uegf, Bmp1) or CCP (complement control protein) domains, which are frequently found in complement system proteins. Proteins that contain these domains are structurally distinct from TARPs (transmembrane AMPA receptor regulatory proteins), and may constitute new protein families that modulate either the localization or function of neurotransmitter receptors. In addition, other CCP domain-containing proteins participate in dendritic patterning and/or synaptic differentiation, although current evidence has not identified any direct activities on neurotransmitter receptors. Some of these proteins are involved in pathologic conditions such as epileptic seizure and mental retardation. Together, these lines of information have shown that CUB and CCP domain-containing proteins contribute to a wide variety of neuronal events that ultimately establish neural circuits.

Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila.

Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch.

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster.

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.

Relationships of Drosophila melanogaster RECQ5/QE to cell-cycle progression and DNA damage.

Members of the RecQ family of DNA helicases are involved in the cellular response to DNA damage and are regulated in the cell-cycle. However, little is known about RecQ5, one of these members. The level of RECQ5/QE, Drosophila melanogaster RecQ5, was increased after the exposure of cultured cells to methyl-methanesulfonate. Transgenic flies that overexpressed RECQ5/QE in their developing eye primordia showed mild roughening of the ommatidial lattice. DNA-damaging agents and the mei-41 mutation enhanced the phenotype caused by RECQ5/QE overexpression. Overexpression of RECQ5/QE perturbed the progression of the cell-cycle in response to DNA damage in the eye imaginal discs. These results suggest that RECQ5/QE interacts with components of the cell-cycle during its progression in response to DNA damage.

Effects of monetary reward and punishment on stimulus-preceding negativity.

This study examined the effects of emotional valence on stimulus-preceding negativity (SPN) using reward and fine. A time estimation task under reward, punishment, combined, and control conditions was performed. Participants were rewarded for accurate responses in the reward condition, and were fined for incorrect estimations in the punishment condition. in the combined condition, correct responses were rewarded and incorrect responses were fined. In the control condition, neither a reward nor fine was used. Results showed a significant interaction of condition x hemisphere. The SPN at the left hemisphere was increased in the reward condition. For the punishment effect, although it evoked right hemisphere dominance, no conditional difference was apparent at the right hemisphere. These results suggest that the SPN is affected by positive emotion: The left hemisphere activation might represent a pleasant emotion accompanying monetary gain.

Selective removal of DNA from protein solution with copolymer particles derived from N,N-dimethylaminopropylacrylamide.

To remove nucleic acids from cellular products as drugs, cross-linked N,N-dimethylaminopropylacrylamide (DMP) particles with cationic functional groups were prepared. The particle's hydrophobicity and its anion-exchange capacity were easily adjusted by changing the cross-linking agent and the DMP ratio in the cross-linking, respectively. When divinylbenzene (DVB) was used as a cross-linking agent and the DMP ratio (in the cross-linking) was adjusted to 90 mol%, the particles (DMP-DVB, 90:10) showed the highest adsorbing activity of DNA (salmon spermary). Its adsorption capacity was 54 mg/ml adsorbent. On the other hand, the adsorption of bovine serum albumin (BSA) to the DMP-DVB extremely increased with increase in the adsorbent's pore size (molecular mass exclusions; M(lim)) from 2 x 10(3) to 1 x 10(4), but decreased with increase in the buffer's ionic strength (mu) to 0.2 or stronger. As a result, when the DMP-DVB (80:20) with M(lim) 2 x 10(3) was used as adsorbent by a column method at pH, 7.2 and mu = 0.17, it only selectively removed DNA from a BSA solution, including 1000 microg/ml of BSA and 10 microg/ml of DNA. The adsorbent decreased the concentration of DNA in the BSA solution to < 10 ng/ml, and the recovery rate of BSA was more 98%.

The possible roles of the DNA helicase and C-terminal domains in RECQ5/QE: complementation study in yeast.

DmRECQ5/QE is a member of the RECQ5 subfamily, which shares homology with the Escherichia coli RecQ DNA helicase. Although the DNA helicase activity of RECQ5/QE has been characterized in vitro, the in vivo function of RECQ5/QE was essentially unknown. To investigate the cellular role of RECQ5, the potential of RECQ5/QE was evaluated by substitution of the only RecQ-like helicase, Sgs1, in budding yeast. RECQ5/QE can complement several phenotypes of sgs1, including the synthetic growth defect with srs2, the hypersensitivity to hydroxyurea and methyl methanesulfonate, and the elevated frequency of homologous recombination and sister chromatid exchange (SCE), but poorly complemented the suppression of slow growth in top3. These data suggested that RECQ5/QE exhibits an evolutionarily conserved RecQ function in vivo. The RECQ5/QE domain necessary for the yeast complementation was determined. The helicase domain and helicase activity were required to complement both the sgs1srs2 and sgs1top3 phenotypes. In contrast, the C-terminal domain was dispensable for complementing the sgs1srs2 phenotype, but was required for the sgs1top3 phenotype. These results suggested that the RECQ5/QE helicase activity is important for cellular function and that the C-terminal domain has a specific function in the absence of Top3.

Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding.

The Drosophila melanogaster RECQ5/QE gene encodes a member of the DNA helicase family comprising the Escherichia coli RecQ protein and products of the human Bloom's, Werner's, and Rothmund-Thomson syndrome genes. The full-length product of RECQ5/QE was expressed in the baculovirus system and was purified. Gel filtration experiments indicated that RECQ5/QE was present in an oligomeric state. The RECQ5/QE protein hydrolyzed ATP and even more actively GTP in the presence of single-stranded DNA. ATP drove the DNA helicase activity of RECQ5/QE, whereas GTP had little effect. GTP exhibited a stimulatory effect on DNA unwinding when it was used together with ATP. This effect was more apparent with non-hydrolyzable GTP analogs, such as GTPgammaS and GMPPNP. These results indicate that GTP binding to RECQ5/QE triggers its DNA helicase activity. GTP binding increased the rate of strand separation without affecting the S(0.5) (K(m)) values for the substrates during the DNA helicase reaction. The data collectively suggest that the RECQ5/QE protein is activated upon GTP binding through the ATP-binding site.