Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei.

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.

Mimosine differentially inhibits DNA replication and cell cycle progression in somatic cells compared to embryonic cells of Xenopus laevis.

The plant amino acid mimosine has been reported to block cell cycle progression and DNA replication in cultured mammalian cells, perhaps by blocking initiation. In this study, we show that mimosine does not block initiation or any other step in DNA replication in embryonic cells of Xenopus laevis. Mimosine does not block DNA replication in cell-free "cycling" extracts of Xenopus eggs, nor does it block M to S phase transition in cell-free egg extracts released from metaphase arrest. Microinjection of mimosine into 4-cell embryos had no visible effect on development during the first 3 days after fertilization. Prior to the midblastula transition, when the cell cycle consists of alternating S and M phases, neither chromosomal DNA replication nor replication of microinjected plasmid DNA were inhibited by mimosine microinjected into cleaving Xenopus embryos. Microinjection of mimosine after the midblastula transition, when large endogenous stockpiles of DNA replication components have begun to be depleted and Xenopus embryonic cells have acquired G1 and G2 phases, still did not inhibit cell cycle progression or DNA replication. In marked contrast, mimosine arrested the growth of proliferating cultured Xenopus kidney epithelial A6 cells near the G1/S boundary. We conclude that mimosine appears to block DNA replication and cell cycle progression in somatic cells, but has no apparent effect in rapidly dividing Xenopus embryonic cells.

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.

Inhibition of DNA replication in cell-free extracts of Xenopus laevis eggs by extracts of Xenopus laevis oocytes.

Cell-free extracts of Xenopus laevis eggs support replication of Xenopus sperm nuclei and purified double-stranded plasmid DNA templates. In contrast, cell-free extracts of Xenopus oocytes do not support replication of these templates. In this study, we show that extracts prepared from Xenopus oocytes, when added to extracts of Xenopus eggs, inhibited replication of Xenopus sperm nuclei and double-stranded plasmid DNA templates. This inhibition did not result from degradation of template DNA by oocyte extracts, nor from inhibition of chain elongation. Nuclei formed in egg extracts in the presence of oocyte extracts were much smaller compared with nuclei formed in egg extracts. Extracts prepared from Xenopus germinal vesicles (oocyte nuclei) also inhibited replication of Xenopus sperm nuclei, but not synthesis of the complementary strand of single-stranded DNA templates in egg extracts. In contrast to extracts prepared from immature oocytes, extracts prepared from Xenopus oocytes matured in vitro effect much less, if any, inhibition of DNA replication. Taken together, these results suggest that molecules that negatively regulate chromosomal DNA replication may be present in Xenopus immature oocytes. The level of these factors may decrease during oocyte maturation, concurrent with the breakdown of oocyte germinal vesicles and manifestation of the capacity of matured oocytes to initiate DNA replication. To our knowledge, this is the first biochemical identification of negative factors that may be involved in the regulation of eukaryotic chromosomal DNA replication.

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.

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.

Molecular analysis of transgenic plants generated by microprojectile bombardment: effect of petunia transformation booster sequence.

Supercoiled plasmid expression vectors containing the petunia transformation booster sequence (TBS) were introduced by microprojectile bombardment into dicotyledenous (tobacco) and monocotyledonous (maize) cells. TBS effected a 7.8- to 16-fold increase in transformation frequencies in tobacco, and a 1.7- to 2.4-fold increase in maize. Although TBS contains a well-defined transcription enhancer element, no increases in plasmid gene expression were observed. TBS did not alter integration patterns in transformants, and did not affect segregation of linkage in R1 progeny. Computer analyses of the TBS sequence revealed numerous modular elements previously shown to be associated with putative chromosomal replication origin regions in eukaryotes, including DNA unwinding elements, scaffold-associated regions and pyrimidine tracts.

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+.

On the nature of origins of DNA replication in eukaryotes.

Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E. coli (oriC) and the tumor virus, SV40 (ori sequence). Initiation events appear to occur throughout broad zones rather than at specific origin sequences. Analysis of four chromosomal origin regions reveals that they share common modular sequence elements. These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase-primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently destabilized regions. Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes. Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single-stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication. DNA synthesis begins subsequently at multiple sites within the unwound regions rather than at specific origin sequences.

Chromosome structures.

Chromosomes are large subcellular structures, visible in the light microscope, that are found in the nuclei of most eukaryotic cells. Each chromosome consists of a single very long DNA molecule that has been compacted approximately 10,000-fold by interactions with proteins, such that the resulting chromosome structure fits within a typical eukaryotic nucleus of only 10 microns in diameter. Several levels of structural organisation are involved in the formation of chromosomes. Most chromosomal DNA is wrapped in left-handed superhelical turns around protein 'spools', called histone octamers, to form nucleosomes. Arrays of these nucleosomes, or 'beads on a string', are further compacted into solenoidal structures, called 30 nm chromatin fibres. The chromatin fibres are, in turn, compacted approximately 250-fold to form topologically independent 'looped' DNA domains, each loop containing about 20,000-100,000 nucleotide pairs of DNA extending from a proteinaceous central scaffold. Some chromosomes, such as lampbrush and polytene chromosomes, can be seen in certain specialised cells during interphase. Metaphase chromosomes, which can be stained to reveal characteristic banding patterns, are formed in most eukaryotic cells during mitosis. Formation of chromosome structures and the nuclei that envelop them involves discrete steps of nucleosome assembly, scaffold assembly, and nuclear envelope assembly, and can be carried out in cell-free extracts of animal eggs. Centromeres, the regions that mediate attachment of a chromosome to a meiotic or mitotic spindle, and telomeres, the natural ends of chromosomes, are structures that ensure that the correct number of full length chromosomes are maintained during the cell cycle. Most chromosome structures (nucleosomes, chromatin fibres, and scaffold loop domains) form from virtually any DNA sequence, but centromeres and telomeres are both composed of specific DNA sequences complexed with specific binding proteins. Recently, complete DNA sequences of entire chromosomes have been determined, and our rapidly emerging knowledge of chromosome structures is beginning to provide insights into the molecular basis of human disease.

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Differential replication of circular DNA molecules co-injected into early Xenopus laevis embryos.

Replication of co-injected supercoiled DNA molecules in fertilized Xenopus eggs was monitored through the blastula stage of development. The extent of replication, as measured by 32P-dTMP incorporation into form I DNA, was directly proportional to the number of molecules, rather than the size, of the plasmid injected. Although only a small fraction of molecules of either template was replicated, incorporation was predominantly into full length daughter molecules. Over at least a 20-fold concentration range of microinjected DNA, injection of equal masses of DNA resulted in greater incorporation into the smaller form I DNA present in molar excess. The extent of incorporation into supercoiled DNA for a particular plasmid was apparently independent of the concentration of a second, co-injected plasmid. The relative extents of replication of co-injected supercoiled templates could be altered simply by changing the molar ratios of the templates.

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase.

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.

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.

Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo.

Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy-nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described.

High-resolution chromatography of nucleic acids on the Gen-Pak FAX column.

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis.

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro.

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

Characterization of a stable, major DNA polymerase alpha species devoid of DNA primase activity.

We have purified from Xenopus laevis ovaries a major DNA polymerase alpha species that lacked DNA primase activity. This primase-devoid DNA polymerase alpha species exhibited the same sensitivity as the DNA polymerase DNA primase alpha to BuAdATP and BuPdGTP, nucleotide analogs capable of distinguishing between DNA polymerase delta and DNA polymerase DNA primase alpha. The primase-devoid DNA polymerase alpha species also lacked significant nuclease activity indicative of the alpha-like (rather than delta-like) nature of the DNA polymerase. Using a poly(dT) template, the primase-devoid DNA polymerase alpha species elongated an oligo(rA10) primer up to 51-fold more effectively than an oligo(dA10) primer. In direct contrast, the DNA polymerase DNA primase alpha complex showed only a 4.6-fold preference for oligoribonucleotide primers at the same template/primer ratio. The catalytic differences between the two DNA polymerase alpha species were most dramatic at a template/primer ratio of 300. The primase-devoid DNA polymerase alpha species was found at high levels throughout oocyte and embryonic development. This suggests that the primase-devoid DNA polymerase alpha species could play a physiological role during DNA chain elongation in vivo, even if it is chemically related to DNA polymerase DNA primase alpha.