DNA unwinding protein from meiotic cells of Lilium.

An ATP-dependent DNA unwinding protein is present at a high level of activity in meiotic cells of lilies. The protein also acts as a DNA-dependent ATPase, the single strand form being the preferred cofactor. It binds in the absence of ATP to single-strand DNA and to ends or nicks in duplex DNA. A 3'-OH terminus is required for binding at duplex ends; such binding is highly stable. Unwinding occurs in the presence of ATP, and it is limited to about 50 base pairs per end or 400-500 base pairs per nick. The ATP hydrolyzed during unwinding is distinguishable from ATP hydrolysis in the presence of single-strand DNA.     

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.     

Activation of phosphatidylinositol transfer protein alpha and beta isoforms from inclusion bodies

Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TPalpha was achieved in the presence of DiC7:0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles. Replacement of DiC7:0-PtdCho with the natural ligands of PI-TPalpha, i.e. long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TPalpha required a low temperature (4 degrees C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 microg ml(-1)). The inclusion bodies yielded 10 mg homogeneous PI-TPalpha per liter of E. coli culture. Conditions for full activation of PI-TPbeta were similar to those for PI-TPalpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 microg ml(-1)) were required. In contrast to PI-TPalpha, PI-TPbeta lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of beta-alanine. In summary, despite 94% sequence similarity, PI-TPalpha and PI-TPbeta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.     

Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha.

DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Genetic selection of meiotic and mitotic recombinant yeast artificial chromosomes.

We have developed a genetic screen for the isolation of larger or smaller recombinant yeast artificial chromosomes derived from overlapping YACs. Integration plasmids were used to modify the TRP1 and URA3 auxotrophic markers present respectively on the left and right vector arms of one of the parental YACs. Diploids containing the two parental YACs were studied through meiosis and mitosis. Tetrad analysis revealed the presence of meiotic recombinant YACs at a frequency comparable with what is expected for yeast DNA (about 3 kb/cM). More direct genetic selection of diploids on -TRP-LYS synthetic media in the presence of 5-fluoro-orotic acid (5-FOA), led to the isolation of mitotic recombinant YACs at a high frequency. Analysis of these yeast cells by pulsed-field gel electrophoresis, confirmed the loss of both parental artificial chromosomes, and the specific retention of a larger or smaller recombinant YAC.     

Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae

The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

Post-translational modifications in mitotic yeast cells

We have recently shown that secretion of invertase is not inhibited in the yeast Saccharomyces cerevisiae during mitosis, but continues, as during interphase. This is in contrast with the mammalian cell, where membrane traffic stops at the onset of prometaphase. Here we extend our findings by showing that the bulk of the cell surface glycoproteins and mannans, as well as the yeast pheromone alpha-factor, traverse the secretory pathway during mitosis. We show that the mitotic cells are able to carry out several types of post-translational modification of secretory proteins. (a) The secretory protein invertase was oligomerized and extensively glycosylated, (b) the N-glycan cores of bulk-cell surface mannans were extended with outer chains, (c) some N-glycans were phosphorylated, (d) the protein-bound O-glycans were extended up to tetramannosides, (e) prepro-ka-factor was proteolytically processed to alpha-factor molecules. We conclude that the secretory pathway in yeast remains fully functional throughout the cell cycle.     

DNA shuffling method for generating estrogen receptor alpha and beta chimeras in yeast

To facilitate our study of the molecular basis for the estrogen receptor (ER) subtype selectivity of novel ligands, we used DNA shuffling to construct chimeric ERs having ligand binding domains derived from both ER alpha and ER beta. The efficiency of chimera generation was low with traditional DNA shuffling protocols. Furthermore, ER ligand binding domain sequences lack convenient restriction sites for introducing chimeric ligand binding domain sequences into expression vectors. To overcome these problems, we developed a modified strategy whereby chimeric sequences were exclusively amplified from among the reassembled products from DNA shuffling using a special pair of PCR primers whose 3' ends specifically match the alpha and beta sequences, respectively, and whose 5' ends match sequences outside the ER beta ligand binding domain. When chimeric ligand binding domain DNA sequences, amplified with these primers, were co-transformed into a yeast strain with a linearized expression vector for ER beta, an active expression vector was produced by homologous recombination. Twenty-two different crossover sites were found; most occurred when there was a stretch of eight or more identical base pairs in both sequences, and many were concentrated in the regions important for studying ligand binding and transactivation. This method should prove to be useful for generating chimeric gene products from parent templates that share relatively low sequence identity.     

Intermediates of yeast meiotic recombination contain heteroduplex DNA

The formation of heteroduplex DNA features prominently in all models for homologous recombination. A central intermediate in the current double-strand break repair model contains two Holliday junctions flanking a region of heteroduplex DNA. Studies of yeast meiosis have identified such intermediates but failed to detect associated heteroduplex DNA. We show here that these intermediates contain heteroduplex DNA, providing an important validation of the double-strand break repair model. However, we also detect intermediates where both Holliday junctions are to one side of the initiating DSB site, while the intervening region shows no evidence of heteroduplex DNA. Such structures are not easily accommodated by the canonical version of the double-strand break repair model.     

Temporal and spatial localization of novel nuclear protein NP95 in mitotic and meiotic cells

Expression of novel NP95 (nuclear protein, 95 kDa), which contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-cyclin-dependent protein kinase 2 phosphorylation site, and retinoblastoma protein-binding motifs, is associated with S-phase progression of mouse cells. It is suppressed during G1 and G2/M phases in normal thymocytes but expressed at a constantly high level irrespective of cell stage in mouse T cell lymphoma cells. NP95 was shown previously to be expressed strongly only in proliferative tissues and cells. In this immunohistochemical study, we demonstrate that NP95 is localized in S-phase nuclei as dot-like foci. Double immunostaining of NP95 and proliferating cell nuclear antigen (PCNA) showed that NP95 was co-localized with PCNA. Construction of three-dimensional images indicated that NP95 was localized with PCNA in replication sites in a somewhat distinct temporal manner. During meiosis, NP95 was present not only in proliferating spermatogonia but also in meiotic spermatocytes and differentiating spermatids which were not proliferating. The possible role of NP95 in mitotic and meiotic cells is discussed.     

Characterization of protein kinases in mitotic and meiotic cell extracts

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Dissociation of meiotic and mitotic roles of the fission yeast cdc2 gene

Summary The fission yeastcdc2 gene is pleiotropic, functioning both in the cell division cycle and in meiosis. Here we show thatcdc2 is allelic totws1, a previously isolated meiotic gene. Dissociation of meiotic and mitotic roles of the gene is also demonstrated by finding mutant alleles specifically altered in only one of the two processes.     

Heterologously expressed polypeptide from the yeast meiotic gene HOP1 binds preferentially to yeast DNA.

HOP1 protein, present in sporulating cells of Saccharomyces cerevisiae and believed to be a component of the synaptonemal complex, has been expressed in Escherichia coli fused to a biotinylated tag protein. Once solubilized from bacterial inclusion bodies, the HOP1 fusion protein was purified by using a combination of avidin-affinity chromatography and gel filtration FPLC and refolded. Sequence comparisons indicate that the HOP1 gene product contains a zinc finger motif, which may confer DNA binding properties, and the recombinant polypeptide was used to assess the putative DNA binding properties of the product of native HOP1 protein using a gel-shift assay. Protein and protein-DNA complexes were detected by exploiting the affinity of streptavidin-alkaline phosphatase for the biotinylated tag protein after Western blotting. The HOP1 fusion protein bound unambiguously to digested genomic yeast DNA. This binding possessed some degree of specificity, was maintained under a wide range of salt concentrations, and was unaffected by the presence of high concentrations of competitor DNA (synthetic poly[dI-dC].poly[dI-dC]). In contrast, no shift was detected when the fusion protein was incubated with digested genomic DNA from Arabidopsis, or with lambda/HindIII DNA. Incubation with digested genomic DNA from Lilium produced a small change in the mobility of the protein. The biotinylated tag protein failed to show any DNA binding activity. Scatchard analysis indicated an apparent yeast genomic DNA:HOP1 fusion protein dissociation constant of K(d) = 5 x 10(-7) M.     

recA protein promoted DNA strand exchange

recA protein and circular single-stranded DNA form a stable complex in the presence of single-stranded DNA binding protein (SSB), in which one recA protein monomer is bound per two nucleotides of DNA. These complexes are kinetically significant intermediates in the exchange of strands between the single-stranded DNA and an homologous linear duplex. After completion of strand exchange, the recA protein remains tightly associated with the circular duplex product of the reaction and the SSB is bound to the displaced linear single strand. Upon addition of ADP, the recA protein-duplex DNA complex dissociates. RecA protein also interacts with single-stranded DNA in the absence of SSB; however, the amount of recA protein bound is substantially reduced. These findings provide direct physical evidence for the participation of SSB in the formation of the recA protein-single-stranded DNA complexes inferred earlier from kinetic analysis. Moreover, they confirm the ability of recA protein to equilibrate between bound and free forms in the absence of SSB.     

Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states

DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. alpha-Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE-cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, alpha- and beta-polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late-passage and postconfluent early passage fibroblasts, amounts of alpha-polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The alpha-polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCI and could be inhibited by a monoclonal IgG fraction prepared against KB cell alpha-polymerase. By several criteria, there was no significant differences in levels of UV-stimulated repair synthesis observed in early or late-passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of alpha-polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for alpha-polymerase from late-passage cells may be attributed more generally to the nondividing nature of these cells.