DEAD-box Protein Research Articles

Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster.

Notch signaling is an evolutionary conserved process that influences cell fate determination, cell proliferation, and cell death in a context-dependent manner. Notch signaling is fine-tuned at multiple levels and misregulation of Notch has been implicated in a variety of human diseases. We have characterized maheshvara (mahe), a novel gene in Drosophila melanogaster that encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. A dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe in a wide range of tissues during development results in a variety of defects, many of which resemble a typical Notch loss-of-function phenotype. We illustrate that ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch-mediated proliferation of eye tissue. Further, we illustrate that mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Coexpression of Deltex and Mahe at the dorso-ventral boundary results in a wing-nicking phenotype and a more pronounced loss of Notch target Cut. Taken together we report identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signaling.

Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein.

The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein-protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein-protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato.

The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants.

Retinoic acid-inducible gene 1 and sensing of hepatitis B virus revisited.

Retinoic acid-inducible gene 1 and sensing of hepatitis B virus revisited.

RNA degradosomes in bacteria and chloroplasts: classification, distribution and evolution of RNase E homologs.

Ribonuclease E (RNase E) of Escherichia coli, which is the founding member of a widespread family of proteins in bacteria and chloroplasts, is a fascinating enzyme that still has not revealed all its secrets. RNase E is an essential single-strand specific endoribonuclease that is involved in the processing and degradation of nearly every transcript in E. coli. A striking enzymatic property is a preference for substrates with a 5' monophosphate end although recent work explains how RNase E can overcome the protection afforded by the 5' triphosphate end of a primary transcript. Other features of E. coli RNase E include its interaction with enzymes involved in RNA degradation to form the multienzyme RNA degradosome and its localization to the inner cytoplasmic membrane. The N-terminal catalytic core of the RNase E protomer associates to form a tetrameric holoenzyme. Each RNase E protomer has a large C-terminal intrinsically disordered (ID) noncatalytic region that contains sites for interactions with protein components of the RNA degradosome as well as RNA and phospholipid bilayers. In this review, RNase E homologs have been classified into five types based on their primary structure. A recent analysis has shown that type I RNase E in the γ-proteobacteria forms an orthologous group of proteins that has been inherited vertically. The RNase E catalytic core and a large ID noncatalytic region containing an RNA binding motif and a membrane targeting sequence are universally conserved features of these orthologs. Although the ID noncatalytic region has low composition and sequence complexity, it is possible to map microdomains, which are short linear motifs that are sites of interaction with protein and other ligands. Throughout bacteria, the composition of the multienzyme RNA degradosome varies with species, but interactions with exoribonucleases (PNPase, RNase R), glycolytic enzymes (enolase, aconitase) and RNA helicases (DEAD-box proteins, Rho) are common. Plasticity in RNA degradosome composition is due to rapid evolution of RNase E microdomains. Characterization of the RNase E-PNPase interaction in α-proteobacteria, γ-proteobacteria and cyanobacteria suggests that it arose independently several times during evolution, thus conferring an advantage in control and coordination of RNA processing and degradation.

Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue

VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5′-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3′-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3′-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P<0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.

When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

CIKS/DDX3X interaction controls the stability of the Zc3h12a mRNA induced by IL-17.

IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17-induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17-induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17-dependent mechanism regulating the stabilization of a selected mRNA.

팔딱이 지렁이(Perionyx excavatus) DDX3 유전자의 동정 및 특성

Helicase는 NTP 결합의 화학적 에너지를 이용하여 이중가닥의 DNA와 RNA를 단일가닥으로 분해하여 다양한 생체반응에 기여하는 단백질로 알려져 있으며, 이 중 DEAD-box의 단백질은 주로 RNA와 관련된 대부분의 생화학적 반응에 작용하는 ATP 의존성 helicase로 알려져 있다. 또한 이 단백질 부류에 속하는 DEAD-box3 (DDX3) gene은 척추동물뿐만 아니라 무척추동물에서의 유성 생식과 무성 생식에서 생식세포 발달 및 재생과정 중 줄기세포 분화에 중요한 역할을 하는 인자로 알려져 있다. 이에 본 연구는 강한 재생능력을 가진 것으로 알려져 있는 팔딱이 지렁이(Perionyx excavatus)에서 DDX3 gene을 동정하고 그 발현양상을 알아보고자 환대를 포함하는 성체 지렁이의 두부를 절단하여 total RNA를 추출하고, 이를 주형으로 RT-PCR을 수행하여 full length의 DDX3 gene인 Pe-DDX3를 검출하였다. Pe-DDX3는 607개 아미노산 서열로 이루어져 있으며, DEAD-box 단백질 그룹 내에서 특이적으로 보존되어 있는 9개의 motif가 존재하고 있다. 다른 분류군에 속하는 동물들과의 multiple alignment를 통해 서열 내에 보존되어 있는 아미노산 서열을 확인할 수 있었으며, 아미노산 차원에서의 계통수 분석을 통해 DDX3 (PL10) 하부그룹에 속하는 것을 알 수 있었으며, 또한, 같은 그룹에 속하는 동물 중 P. dumerilii의 PL10a, b 단백질과 가장 가까운 유연관계를 확인 할 수 있었다. Helicases are known to be a proteins that use the chemical energy of NTP binding and hydrolyze to separate the complementary strands of double-stranded nucleic acids to single-stranded nucleic acids. They participate in various cellular metabolism in many organisms. DEAD-box proteins are ATP-dependent RNA helicase that participate in all biochemical steps involving RNA. DEAD-box3 (DDX3) gene is belonging to the DEAD-box family and plays an important role in germ cell development in many organisms including not only vertebrate, but also invertebrate during asexual and sexual reproduction and participates in stem cell differentiation during regeneration. In this study, in order to identify and characterize DDX3 gene in the earthworm, Perionyx excavatus having a powerful regeneration capacity, total RNA was isolated from adult head containing clitellum. Full length of DDX3 gene from P. excavatus, Pe-DDX3, was identified by RT-PCR using the total RNA from head as a template. Pe-DDX3 encoded a putative protein of 607 amino acids and it also has the nine conserved motifs of DEAD-box family, which is characteristic of DEAD-box protein family. It was confirmed that Pe-DDX3 has the nine conserved motifs by the comparison of entire amino acids sequence of Pe-DDX3 with other species of different taxa. Phylogenetic analysis revealed that Pe-DDX3 belongs to a DDX3 (PL10) subgroup of DEAD-box protein family. And it displayed a high homology with PL10a, b from P. dumerilii.

Unwinding the Mechanisms of a DEAD-Box RNA Helicase in Cancer

RNA helicases function in all organisms to manage the conformational states of RNAs of all stripes. The largest family of RNA helicases is the DEAD-box family, with 37 members in humans [1]. DEAD-box proteins interact with and manipulate RNAs that range from rRNAs, as they fold and assemble into ribosomes, to mRNAs as they are exported from the nucleus, prepared for translation, and eventually destroyed. One of the best-studied and versatile DEAD-box proteins is DDX3X (Ded1 in yeast), which is implicated in processes including transcription, mRNA export, translation, storage, and decay [2–4].

Learning from ribozymes

Learning from ribozymes

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.

Cytoplasmic hGle1A regulates stress granules by modulation of translation.

When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA-mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.

Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1′s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an ‘open’ to a ‘closed’-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.

The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly

Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process, and factors involved remain largely unknown. Here, we have identified the DEAD-box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability or 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis, and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that takes place mainly near the mitochondrial nucleoids, in the compartment defined by the RNA granules.

RNA helicase DP103 and TAK1: a new connection in cancer

Article on new markers of tumor metastasis. The authors found that expression of DP103 was significantly upregulated in the metastatic basal subtype of breast cancers in 3 independent cohorts. This elevated DP103 expression correlated with metastatic breast cancer gene signatures and was strongly associated with patient survival. This study provides the first evidence that the RNA helicase DEAD-box protein DP103 is an NF-kB target that could form part of a positive feedback loop contributing to DP103- mediated regulation of TAK1 kinase activity on the major NF-kB kinase IKK2, thus implicating DP103 in the maintenance of this oncogenic signaling arm in human cancer.

Kinetics and Thermodynamics of the ATPase Cycle of the DEAD-Box Protein Dbp5

DEAD-box proteins are ubiquitous ATPase motor proteins involved in all aspects of RNA metabolism including folding, duplex unwinding and ribonucleoprotein remodeling. Although all characterized DEAD-box proteins share a similar ATPase cycle, differences in individual ATP utilization rate and equilibrium constants confer unique motor properties for carrying out physiological functions, analogous to cytoskeletal motors (i.e. myosin, kinesin, dynein). RNA helicase functional diversity is therefore contained in their ATPase cycles, and versatility introduced through interactions with regulatory partners. Dbp5 is a yeast DEAD-box protein that, in conjunction with several regulatory proteins and small molecules, plays an essential role in mRNA export from the nucleus to cytosol. We present a kinetic and equilibrium analysis of the Dbp5 ATPase as context for studying the role of the regulatory partners.

Auxiliary Factors and RNA Substrates Regulate Dead-Box Protein Activity by Modulation of the Dead-Box Protein Conformational Cycle

DEAD-box proteins unwind RNA duplexes at the expense of ATP hydrolysis in virtually all processes involving RNA. During unwinding, they alternate between open and closed conformations, and the transition to the closed conformation has previously been linked to duplex destabilization. The eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box helicase that is thought to resolve secondary structure elements from the 5′-UTR of mRNAs to enable ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. eIF4A can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by eIF4G, and a closed state populated in the presence of eIF4G and eIF4B. In single molecule FRET experiments on donor/acceptor-labeled eIF4A, we have dissected the effect of eIF4B and eIF4G on RNA-dependent ATPase- and RNA helicase activities, and on the eIF4A conformational cycle in the context of different unwinding substrates. We show that eIF4B and eIF4G, as well as different structures in the 5′-UTR, modulate the energy landscape underlying the eIF4A conformational cycle by changing the energetic differences and the energy barriers between functionally relevant conformational states, leading to changes in rate constants for inter-conversion and in equilibrium distributions. Our results reveal on a molecular level how translation initiation factors synergistically stimulate the eIF4A helicase activity in the mRNA scanning process, and show that the eIF4A conformational cycle is central for the multi-layered regulation of eIF4A activity, and for its role as a regulatory hub in translation initiation.

U2 snRNA Conformation is Regulated by Cus2 to Facilitate Dead-Box Protein Loading

The spliceosome undergoes dynamic changes in composition and conformation during assembly and splicing. We are interested in studying how the spliceosomal U2 snRNP loads onto pre-mRNA branchsites during assembly. This involves multiple conformations of the U2 snRNA (stem IIa vs. stem IIc), the Prp5 DEAD-box protein, and the Cus2 RRM protein. Our results show that Cus2 binds to WT or stem IIa stabilized model RNAs but not to stem IIc. In contrast, Prp5 binds indiscriminately to all three types of U2 RNA and each equally stimulates Prp5's ATPase. Prp5 appears to either displace or out compete Cus2 for U2 RNA binding in an ATP-independent process. Using single molecule FRET, we show the U2 core RNA is dynamic with interconverting populations of high- (likely stem IIc) and mid-FRET states (likely stem IIa). Addition of Cus2 depletes the high-FRET state, consistent with conformational selection of U2 stem IIa by Cus2. Together, our data support spliceosomal proteins acting in concert to facilitate loading of the DEAD-box onto a particular RNA structure. This represents a new paradigm for protein:RNA interactions that is distinct from ordered binding of structure specific RNA-binding proteins (i.e., ribosome assembly) or from positional control of DEAD-box protein loading during exon junction complex formation.

Single-Molecule Study of Ded1 Helicases using a Hairpin Substrate

DEAD-box helicases remodel RNA structures, DNA/RNA hybrids and RNA-protein complexes that are essential in gene regulation. So far their mechanism and functioning is very much debated and the classical or bulk assays are not sufficient to answer these questions. We have developed a new simple magnetic tweezers based single molecule cyclic assay, acting in parallel on tens of molecules at the same time, which allows detecting the unwinding of short RNA/DNA hybrids. Comparing this information with and without an interacting agent can present some insights about the agent. We implement our assay to study DEAD-box helicase Ded1 which is known to only melt a few bases of DNA/RNA duplex. The observation of unwinding by Ded1 helicase in the presence of ATP indicates that the enzyme melt the duplex rather than translocating along ssDNA. Although DEAD-box proteins are most active with substrates containing single-stranded overhangs, they are also able to unwind short blunt-end duplexes. This has led to the proposal that DEAD-box proteins directly interact with the duplex, but they are “activated” by single-stranded RNA bound at another site. Moreover, it has been proposed that the proteins work by localized destabilization of the RNA helical regions. Hence, the proteins lack processivity. We performed the helicase activity measurement at various concentrations of proteins. Our results show that Ded1 activity is maximum in case of RNA oligo with 5’ overhang. The helicase activity is observed only in presence of ATP while the ATP analogues supported annealing activity. We have also confirmed that the enzyme has no visible effect on the unwinding of DNA.