Laevis DNA Research Articles

EDNA is a useful tool to evaluate the success of the eradication program of Xenopus laevis in Portugal

Biological invasions are widely recognized as a major driver of global biodiversity loss. The most cost-effective answer is often population eradication, while the number of individuals is still limited. The detection of the invasive species at low densities is essential for eradication success, which can be difficult using traditional methods. Environmental DNA (eDNA) can facilitate the detection and monitoring of invasive aquatic species at low densities and can be more sensitive than traditional sampling. The African clawed frog (Xenopus laevis) is a highly invasive species and has been recorded in several European countries. In Portugal, the species was discovered in 2006 and since 2010, streams of the Lisbon area, suspected or at risk of invasion, are being monitored every year and removal is ongoing. The main method of detection is electrofishing but methods like draining artificial ponds have also been used. This program so far succeeded in containing the spread and reduced frog abundance, although total eradication has not yet been accomplished. To evaluate the success of this program, we collected water samples from fifteen sites. In each site we sampled both pools and riifles to test if lotic microhabitat influences X. laevis eDNA detection and concentration. Surface water velocity in each sampling point was also measured. We filtered the samples, extracted DNA from filters, and assayed the extracted DNA for X. laevis DNA using quantitative polymerase chain reaction (qPCR). From the fifteen sites sampled, five were positive for X. laevis eDNA: four lotic sites and one lentic site. Local eradication success was evident mainly in lentic habitats, and three potential failures were also identified. Finding X. laevis at one of these sites only after X. laevis eDNA detection was an important contribution of this technique for the success of the control and eradication program. No significative differences were found between lotic microhabitat (riffles and pools) regarding X. laevis eDNA concentration and detection but velocity affected the concentration of X. laevis eDNA captured. Our results corroborate other studies that recommend eDNA as a complementary sampling approach to other traditional methods and suggests its suitability as a tool for the detection of invasive amphibians. To be presented as a flash oral presentation at the DNAQUA International Conference - First international conference on the use of DNA for water biomonitoring.

Development of ISSR-derived SCAR Marker and SYBR Green I Real-time PCR Method for Detection of Teliospores of Tilletia laevis K\xfchn

Common bunt, caused by Tilletia laevis Kühn [syn. T. foetida (Wallr) Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.

Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization

We examined the composition of telomeres in chromosomes of parasitic worms, representatives of the flatworm groups Monogenea and Cestoda (Platyhelminthes), and thorny-headed worms (Syndermata: Acanthocephala) by fluorescence in situ hybridization (FISH) with different telomeric repeat probes. Our results show that the (TTAGGG)n sequence, supposed to be the ancestral telomeric repeat motif of Metazoa, is conserved in Monogenea (Paradiplozoon homoion) and Cestoda (Caryophyllaeus laticeps, Caryophyllaeides fennica, and Nippotaenia mogurndae) but not in Acanthocephala (Pomphorhynchus laevis and Pomphorhynchus tereticollis). In the Pomphorhynchus species, no hybridization signals were obtained with the "nematode" (TTAGGC)n, "arthropod" (TTAGG)n, and bdelloid (TGTGGG)n telomeric probes using FISH with their chromosomes and Southern hybridization with P. laevis DNA. Therefore, we suggest that parasitic Acanthocephala have evolved yet unknown telomeric repeat motifs or different mechanisms of telomere maintenance.

Xenbase: a Xenopus biology and genomics resource

Xenbase (www.xenbase.org) is a model organism database integrating a diverse array of biological and genomic data on the frogs, Xenopus laevis and Xenopus (Silurana) tropicalis. Data is collected from other databases, high-throughput screens and the scientific literature and integrated into a number of database modules covering subjects such as community, literature, gene and genomic analysis. Gene pages are automatically assembled from data piped from the Entrez Gene, Gurdon Institute, JGI, Metazome, MGI, OMIM, PubMed, Unigene, Zfin, commercial suppliers and others. These data are then supplemented with in-house annotation. Xenbase has implemented the Gbrowse genome browser and also provides a BLAST service that allows users to specifically search either laevis or tropicalis DNA or protein targets. A table of Xenopus gene synonyms has been implemented and allows the genome, genes, publications and high-throughput gene expression data to be seamlessly integrated with other Xenopus data and to external database resources, making the wealth of developmental and functional data from the frog available to the broader research community.

Isolation and characterization of microsatellite loci from the Australasian sea snake, Aipysurus laevis

AbstractWe developed 13 microsatellite loci for the olive sea snake, Aipysurus laevis, using both enriched and unenriched genomic DNA libraries. Eleven codominant loci, that reliably amplified, were used to screen 32 individuals across the geographic range of A. laevis. Four loci had four or more alleles (maximum 12), whereas the other seven had either two or three. All but one locus was in Hardy–Weinberg equilibrium. These loci will provide useful markers to investigate population genetic structure for the olive sea snake.

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.

A Tribute to the Xenopus laevis Oocyte and Egg

When I was asked to reflect as part of the celebration of the 100-year anniversary of the Journal of Biological Chemistry I recalled a talk by Seymour Cohen at the Federation meetings in Atlantic City about 1960. He began by thanking bacteriophage for providing him with so many wonderful research problems. I decided in the same spirit to pay homage to the Xenopus laevis egg and oocyte, two states of a cell that has played and continues to play a central role in most of the disciplines of modern biology including biochemistry and molecular biology. Many of us owe a debt of gratitude to this cell. The egg is the most important and interesting cell in the repertoire of any organism. In X. laevis this single cell has a diameter of about 1.3 mm. The oocyte is permeable to small molecules, but after it undergoes meiosis and becomes an unfertilized egg it is impermeable to these same molecules. The oocyte is an active site of RNA and protein synthesis but not DNA replication, whereas the unfertilized egg is poised to replicate every 10 min after it is fertilized. During the cleavage period the embryo is transcriptionally silent. An oocyte can be cultured for days, but once ovulated the egg degenerates rapidly if it is not fertilized. The oocyte is the largest single cell in the body yet it has many of the same structures as somatic cells. These compartments are so exaggerated in size that they are accessible to cell biologists for manipulation and visualization. The huge oocyte nucleus is called a germinal vesicle (GV). The premeiotic tetraploid chromosomes are expanded into a “lampbrush” configuration so that they can be studied with a light microscope. The X. laevis egg and its possibilities for experimental manipulation were introduced to modern biology in the late 1950s by John Gurdon (Fig. 1), then a graduate student in the laboratory of Michail Fischberg in the Department of Zoology at Oxford University. Gurdon’s thesis was to reproduce with the South African “clawed toad” X. laevis the famous nuclear transplantation experiments that Briggs and King had perfected with the American leopard frog, Rana pipiens (1). Embryonic nuclei were injected into eggs whose own nuclei had been removed or destroyed. These early cloning experiments addressed the question of whether a differentiated and specialized somatic cell nucleus was still capable of expressing its full genetic repertoire. Briggs and King found that nuclei from R. pipiens embryos older than gastrula never promoted normal development, suggesting that the genetic material might change in cells as they specialize during embryonic development. This result was challenged by John Gurdon’s demonstration that nuclei from X. laevis embryos are more permissive. Even a small fraction of nuclei derived from differentiated intestinal epithelial cells supported normal development (2). This crucial scientific question of whether irreversible changes occur in the genome of highly specialized cells was addressed more precisely by experimental manipulation than by biochemistry or genetics. The concepts behind these experiments have influenced the stem cell field. The topic of whether the genome is altered in specialized somatic cells was revisited just this year by the same strategy of nuclear transplantation. Postmitotic nuclei from olfactory neurons support the development of normal fertile mice when transplanted into enucleated mouse eggs (3). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45291–45299, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Isolation and expression of a Xenopus laevis DNA methyltransferase cDNA.

A Xenopus DNA methyltransferase cDNA was isolated from a Xenopus oocyte cDNA library by screening with the mouse DNA methyltransferase cDNA as a probe. The elucidated nucleotide sequence gave a 4,470 nucleotide open reading frame, and the predicted protein was composed of 1,490 amino acid residues, showing high homology to animal DNA methyltransferases, especially in the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and the Lys-Gly repeat which were first found in the mouse sequence were conserved in Xenopus. However, 200 amino acid residues at the amino-terminus of Xenopus DNA methyltransferase were quite different from those of mouse and human, but showed 70% homology with those of chicken. The cloned Xenopus DNA methyltransferase cDNA expressed in COS1 cells showed a significant DNA methyltransferase activity. The size of the translation product of Xenopus DNA methyltransferase cDNA expressed in COS1 cells was identical with that of the endogenous DNA methyltransferase in Xenopus A6 cells and also with the size of newly synthesized DNA methyltransferase in Xenopus oocytes. However, a slightly larger immunoreactive band of about 205 kDa, and a small immunoreactive band of about 100 kDa, which were poorly labeled by short incubation with radiolabeled amino acids, were the main bands in stage I-III and stage IV-VI oocytes, respectively.

Termination within Oligo(dT) Tracts in Template DNA by DNA Polymerase γ Occurs with Formation of a DNA Triplex Structure and Is Relieved by Mitochondrial Single-stranded DNA-binding Protein

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.

Association of rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization.

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70-160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0-1.4 micrograms/10(7) sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25-30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed.

The cloning and characterization of a cDNA encoding Xenopus laevis DNA ligase I

A cDNA clone coding for DNA ligase I (LigI) was isolated from a Xenopus laevis oocyte cDNA library. The 3766-bp sequence showed a putative ORF capable of encoding a 1070-amino-acid protein whose overall identity with two mammalian sequences is 63%. This identity, however, rises to 72.5% in the C-terminal portion of the protein that contains the active site. Expression of the cDNA in a prokaryotic system produces a protein that is immunologically identical to LigI and can be adenylated. The 180-kDa size of the recombinant protein is similar to the LigI detected in oocyte. Northern blot analysis of ovary and embryo RNAs revealed the expression of two (4.1 and 6 kb) LigI transcripts.

Action of Mitochondrial DNA Polymerase γ at Sites of Base Loss or Oxidative Damage

Mitochondrial DNA is subject to oxidative damage generating 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) residues and to spontaneous or induced base loss generating abasic sites. Synthetic oligonucleotides containing these lesions were prepared and used as templates to determine their effects on the action of Xenopus laevis DNA polymerase gamma. An analogue of an abasic site in DNA, tetrahydrofuran, was found to inhibit elongation by DNA polymerase gamma. When the DNA polymerase was able to complete translesional synthesis, a dA residue was incorporated opposite the abasic site. In contrast, elongation by DNA polymerase gamma was not inhibited by an 8-oxo-dG residue in the template strand. The polymerase inserted dA opposite 8-oxo-dG in approximately 27% of the extended products. The effects of these lesions on the 3'-->5' exonuclease proofreading activity of DNA polymerase gamma were also investigated. The 3'-->5' exonuclease activity excised any of the four normal bases positioned opposite either a tetrahydrofuran residue or 8-oxo-dG, suggesting that proofreading may not play a major role in avoiding misincorporation at abasic sites or 8-oxo-dG residues in the template. Thus, both of these lesions have the prospect of causing high rates of mutation during mtDNA replication.

Casein kinase II stimulates Xenopus laevis DNA topoisomerase I by physical association.

A Xenopus laevis casein kinase II-like activity copurified with X. laevis DNA topoisomerase I activity during chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite, but the two activities were resolved by chromatography on DNA-agarose [Kaiserman, H. B., Ingebritsen, T. S., & Benbow, R. M. (1988) Biochemistry 27, 3216-3222]. Phosphorylation of the catalytic polypeptides of dephosphorylated X. laevis DNA topoisomerase I by the endogenous X. laevis casein kinase II-like activity apparently resulted in a severalfold increase in catalytic activity. In this study, we show that incubation of purified X. laevis DNA topoisomerase I with electrophoretically homogeneous bovine brain casein kinase II and ATP strongly stimulated catalytic activity. Surprisingly, purified bovine casein kinase II stimulated X. laevis DNA topoisomerase I activity by more than an order of magnitude in the absence of ATP, although ATP resulted in additional stimulation. Other basic proteins, such as histone H1 and HMG proteins, also stimulated X. laevis DNA topoisomerase I catalytic activity 2-3-fold in the absence of ATP. Modulation of catalytic activity by direct physical association (protein-protein interactions) must, therefore, be considered in addition to phosphorylation in assessing the physiological role of casein kinase II and other basic proteins during regulation of X. laevis DNA topoisomerase I activity in vivo.

Xenopus laevis ovarian DNA helicase I: A 3' to 5' helicase that unwinds short duplexes.

A novel DNA helicase isolated from Xenopus laevis ovaries [Poll, E. H. A., & Benbow, R. M. (1988) Biochemistry 27, 8701-8706] was characterized biochemically. The directionality of DNA unwinding was determined to be 3' to 5'. A short 3' ssDNA tail adjacent to duplex DNA was required for DNA unwinding; the minimum length of this tail was between four and nine bases. Only short duplex DNA regions were unwound: duplex DNA of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not. Longer duplex regions were unwound in the presence of Escherichia coli single-strand DNA binding protein if, in addition, the duplex region was flanked by an unpaired 3' or 5' tail and the substrate resembled a branched replicative intermediate. X. laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA. DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 125 mM for Na chi PO4 or K chi PO4. The ATP analog ATP gamma S inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPCP and AMPPNP moderately inhibited DNA unwinding activity and had little effect on the copurifying DNA-dependent ATPase activity. CTP was a relatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dCTP, dGTP, or TTP showed moderate or no inhibition. The copurifying DNA-dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, dGTP, or TTP.

Cloning and expression of cDNA encoding Xenopus laevis bone morphogenetic protein-1 during early embryonic development

The Xenopus laevis DNA fragment encoding a protein homologous with human bone morphogenetic protein-1 (BMP-1) was amplified by polymerase chain reaction (PCR) from cDNA derived from stage 26 (st.26) embryonic RNA. Subsequently this fragment was used as a probe to isolate cDNA clones by screening of a X. laevis st.24 embryonic cDNA library. Two partial clones (22 and 63) were obtained and the missing 5'-end of the clone 22 was extended by the anchored PCR technique. The nucleotide sequence of the resulting clone (22AN) contained an open reading frame coding for a protein with 707 deduced amino acids. Three sizes of mRNA (2.9 5.2 and 6.6 kb) were detected in blastula (st.9) and early gastrula (st.10) embryos, and in hatched tadpole (st.40), but little or no expression was observed in morula (st.7) and late gastrula (st.12) embryos, suggesting a physiological role(s) of X. laevis BMP-1 in normal embryonic development.

An inhibitor of DNA topoisomerase I from Xenopus laevis ovaries

A novel, heat-resistant and Pronase-sensitive, inhibitor of eukaryotic DNA topoisomerase I has been purified from Xenopus laevis ovaries. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the most purified fraction revealed three bands with apparent molecular masses of 25, 28.5, and 33.5 kDa. The 25- and 33.5-kDa peptides recovered from an SDS-PAGE gel inhibited X. laevis DNA topoisomerase I. The purified inhibitor was specific to DNA topoisomerase I and did not inhibit other DNA enzymes tested. The inhibitor blocked the catalytic activity of DNA topoisomerase I by interacting with the enzyme, rather than by competing for binding sites on substrate DNA. Binding of DNA topoisomerase I to substrate DNA was blocked by the inhibitor, as was the cleavage reaction catalyzed by DNA topoisomerase I. Inhibition of DNA topoisomerase I was relieved by divalent cations Ca2+, Mg2+, or Mn2+.

Cloning and nucleotide sequence of a complementary DNA encoding Xenopus laevis metallothionein: mRNA accumulation in response to heavy metals.

A cDNA encoding Xenopus laevis metallothionein (MT) was cloned from a cDNA library constructed using liver poly(A+)RNA of X. laevis adult males treated with CdCl2. The probe used to screen the library was a MT-specific DNA fragment obtained by means of the polymerase chain reaction (PCR) and degenerate oligodeoxynucleotide primers. The cDNA clone encodes a putative protein of 62 amino acids, of which 20 are cysteine residues. The position of all the cysteine residues is conserved with respect to mammalian MT sequences. The amino acid sequence of this X. laevis MT, designated XIMT-A, shares between 60% and 67% identity with various vertebrate MTs. Overall, the structure of XIMT-A is no similar in sequence to MT-1 than it is to MT-2 isoforms of various vertebrates. Ten different X. laevis MT cDNA isolates were partially sequenced and turned out to be identical, suggesting a single species of MT mRNA. Southern blot analysis of X. laevis DNA reveals that the XlMT-A gene is present in at least two copies. This result is consistent with the suggestion that a genome duplication occurred in a X. laevis ancestor. The in vivo response to increasing doses of Cd2+, Zn2+, and Cu2+ metal salts was tested. In the liver, all three metals proved to be potent inducers, raising MT mRNA levels between 50- and 100-fold. The maximum response to Cd2+ was at 12 hr after injection and to Zn2+ at 24 hr after injection. High levels of mRNA were maintained for more than 48 hr. Cd2+ and Zn2+ induced XlMT-A mRNA in all tissues examined (kidney, spleen, heart, intestine, testes, and brain). Dexamethasone did not induce MT mRNA synthesis in the liver.

DNA polymerase γ from Xenopus laevis: I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure

DNA polymerase gamma has been purified over 10,000-fold from mitochondria of Xenopus laevis ovaries. We have developed a novel technique which specifically photolabels DNA polymerases. This procedure, the DNA polymerase trap, was used to identify a catalytic subunit of 140,000 Da from X. laevis DNA polymerase gamma. Additional catalytically active polypeptides of 100,000 and 55,000 Da were identified in the highly purified enzyme. These appear to be products of degradation of the 140,000-Da subunit. The DNA polymerase trap, which does not require large amounts of enzyme or renaturation from sodium dodecyl sulfate, is an alternative to the classic activity gel.

DNA polymerase γ from Xenopus laevis: II. A 3′→5′ exonuclease is tightly associated with the DNA polymerase activity

Abstract Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease. The purified enzyme lacks 5'----3' exonuclease and endonuclease activity. The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures. In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol. The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function. The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.

A DNA helicase from Xenopus laevis ovaries.

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.