Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein.

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.     

Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments.

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.     

Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.

An enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions was partially purified approximately 500- to 1000-fold by DEAE-cellulose chromatography, gel filtration on Sephacryl S300, and chromatography on single-stranded DNA-cellulose. The partially purified enzyme did not have any detectable nuclease activity when tested with single-stranded or double-stranded bacteriophage T7 substrate DNA and did not have detectable endonuclease activity when tested with bacteriophage M13 viral DNA or plasmid pBR322 covalently closed circular DNA. Analysis of the products of the cruciform cleavage reaction by electrophoresis on polyacrylamide gels under denaturing conditions revealed that the cruciform structure was cleaved at either of two sites present in the stem of the cruciform and was not cleaved at the end of the stem. The cruciform cleavage enzyme was able to cleave the Holliday junction present in bacteriophage G4 figure-8 molecules. Eighty percent of these Holliday junctions were cleaved in the proper orientation to generate intact chromosomes during genetic recombination.     

Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae.

Protein extracts from Saccharomyces cerevisiae have been fractionated to reveal a nuclease activity that cleaves cruciform structures in DNA. Negatively supercoiled plasmids that contain inverted repeats that are extruded into cruciform structures have been used as DNA substrates. The sites of cleavage of pColIR215 DNA are located within the extruded cruciform stems and are symmetrically opposed to each other across the cruciform junction. Neither relaxed duplex DNA nor single-stranded DNA serve as substrates. The native molecular weight of the activity was estimated to be approximately equal to 200,000 by gel filtration.     

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

RecT protein of Escherichia coli promotes the formation of joint molecules between homologous linear double-stranded M13mp19 replicative-form bacteriophage DNA and circular single-stranded M13mp19 DNA in the presence of exonuclease VIII, the recE gene product. The joint molecules were formed by a mechanism involving the pairing of the complementary strand of the linear double-stranded DNA substrate with the circular single-stranded DNA substrate coupled with the displacement of the noncomplementary strand. When the homologous linear double-stranded DNA substrate had homologous 3' or 5' single-stranded tails, then RecT promoted homologous pairing and strand exchange in the absence of exonuclease VIII. Histone H1 could substitute for RecT protein; however, joint molecules formed in the presence of histone H1 did not undergo strand exchange. These results indicate that under the reaction conditions used, the observed strand exchange reaction is promoted by RecT and is not the result of spontaneous branch migration. These results are consistent with the observation that expression of RecE (exonuclease VIII) and RecT substitutes for RecA in some recombination reactions in E. coli.     

Purification of tropomyosin from Saccharomyces cerevisiae and identification of related proteins in Schizosaccharomyces and Physarum.

Tropomyosin is a key component of the contractile systems found in muscle and nonmuscle cells of higher eukaryotes. Based on properties common to all tropomyosins, we have purified a protein from Saccharomyces cerevisiae that resembles tropomyosins from higher cells. The yeast protein remains soluble after heat treatment at 90 degrees C, has an apparent polypeptide molecular weight of 33,000, an isoelectric point of 4.5, a Stokes radius of 3.5 nm, and a sedimentation coefficient of 2.6 S. It binds F-actin in a Mg2+-dependent, KCl-modulated manner, up to a stoichiometry of about 1 polypeptide per 3.0 actin monomers. In all these properties it is very similar to tropomyosins from higher cells. Antigen-affinity-purified antibodies specifically recognize the Mr 33,000 polypeptide among total yeast proteins and crossreact with bovine brain tropomyosin. In addition, the antibodies specifically crossreact with heat-stable Mr 33,000 polypeptides in extracts of Schizosaccharomyces pombe and Physarum polycephalum. Our detection of tropomyosin in lower eukaryotes suggests that they might have contractile systems very similar to those found in higher organisms.     

Calf thymus histone H1 is a recombinase that catalyzes ATP-independent DNA strand transfer.

An activity that catalyzes the strand transfer from linear double-stranded tetracycline-resistance gene (tetr) DNA to circular M13mp8-tetr viral DNA was detected in a crude extract from calf thymus. This activity was purified to near, if not complete, homogeneity as judged by NaDodSO4/polyacrylamide gel electrophoresis. We have tentatively named this protein calf thymus strand-transfer protein 1 (CTST1). The apparent molecular mass of the protein was 35 kDa by gel electrophoresis. Its sedimentation coefficient was approximately 1.5 S in glycerol gradient centrifugation. These values led us to examine the possibility that CTST1 is histone H1. Western blot analysis of CTST1 with anti-rat liver histone H1 antiserum showed that CTST1 crossreacts with the serum, indicating that CTST1 is histone H1. The mobility of CTST1 was identical to one of the subtypes of calf thymus histone H1 by NaDodSO4/polyacrylamide gel and acetic acid/urea/polyacrylamide gel electrophoreses. We have also confirmed the above conclusion by showing that calf thymus histone H1 has a strand-transfer activity with a specific activity comparable to that of CTST1. The reaction required homologous substrates, but neither Mg2+ nor ATP. The reaction also required stoichiometric amounts of protein. The purified CTST1 fraction lacked detectable exo- and endonuclease activities and also lacked a DNA helicase activity.     

Isolation and characterization of the gene encoding 2,3-oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae.

The ERG7 gene encoding oxidosqualene-lanosterol cyclase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] from Saccharomyces cerevisiae has been cloned by genetic complementation of a cyclase-deficient erg7 strain. The DNA sequence of this gene has been determined and found to contain an open reading frame of 2196 nt (including stop codon) that encodes a predicted protein of 731 amino acids. The predicted molecular mass of the S. cerevisiae cyclase, 83.4 kDa, is similar to the predicted molecular masses of the oxidosqualene-lanosterol cyclase from Candida albicans and the oxidosqualene-cycloartenol cyclase from Arabidopsis thaliana, as well as to the molecular masses assigned to vertebrate oxidosqualene-lanosterol cyclases; however, it is substantially larger than the molecular mass assigned to purified S. cerevisiae cyclase. At the level of DNA and predicted amino acid sequences, the S. cerevisiae and C. albicans cyclases share 56% and 63% identity, respectively. Tryptophan and tyrosine residues are unusually abundant in the predicted amino acid sequences of (oxido)-squalene cyclases, leading to a hypothesis that electron-rich aromatic side chains from these residues are essential features of cyclase active sites.     

The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity.

The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis. The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58- and 56-kDa proteins, respectively. Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele. To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein. CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1. Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated greater than 10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product. In addition, we show that CDC7 is a phosphoprotein itself. Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.     

Construction of strains of Saccharomyces cerevisiae that grow on lactose.

We have constructed strains of Saccharomyces cerevisiae that grow on lactose (Lac+). S. cerevisiae strain YNN27, which, like all S. cerevisiae, is unable to grow on lactose, was transformed with pKR1B-LAC4-1. This plasmid has a selectable marker gene conferring resistance to the antibiotic G418 and carries a 13-kilobase region of the Kluyveromyces lactis genome including LAC4, a beta-galactosidase gene. Transformants were selected first for G418 resistance and then for growth on lactose. Southern hybridization experiments showed that Lac+ transformants had integrated 15-25 tandem copies of the vector into a host chromosome. Several lines of evidence indicate that the Lac+ phenotype in pKR1B-LAC4-1-transformed S. cerevisiae is due to expression of a K. lactis lactose permease gene that lies between 2 and 8.6 kilobase upstream of LAC4 and also to expression of LAC4. The permease gene has been designated LAC12.     

Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae.

A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2Val19 are mediated through its changes in cAMP concentration.     

ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae.

An activity that catalyzes the transfer of a strand from a duplex linear molecule of DNA to a complementary circular single strand can be detected in crude extracts from mitotic and meiotic cells of the yeast Saccharomyces cerevisiae by adding yeast single-stranded DNA binding proteins. This DNA strand-transfer activity increases greater than 15-fold during meiosis in MATa/MAT alpha diploids prior to the detection of a 100- to 1000-fold increase in homologous chromosomal recombination. No increase is observed in MATa/MATa or MAT alpha/MAT alpha cells, which do not undergo meiosis when shifted to meiotic medium, suggesting the activity is related to meiotic recombination. The activity is named strand-transfer protein alpha (STP alpha) and has been extensively purified from the meiotic cells (6 hr after exposure to sporulation medium). The apparent molecular mass of STP alpha is 38 kDa under denaturing conditions. The DNA strand-transfer reaction catalyzed by STP alpha requires homologous single-stranded and double-stranded DNA and Mg2+ but no nucleotide cofactor. Yeast single-stranded DNA binding proteins stimulate the reaction at least 10-fold. Among the products analyzed by electron microscopy were typical strand-exchange structures.     

Purification and characterization of an alpha-bungarotoxin receptor that forms a functional nicotinic channel.

Neither the structure nor the function of alpha-bungarotoxin (alpha Bgtx) binding molecules in the nervous system have yet been completely defined, although it is known that some of these molecules are related to cation channels and some are not. Using an improved method of affinity chromatography, we have isolated a toxin binding molecule from chicken optic lobe that contains at least three subunits with apparent Mr values of 52,000, 57,000, and 67,000. The Mr 57,000 subunit binds alpha Bgtx and seems to be present in two copies per receptor. The receptor is recognized by antibodies raised against the alpha Bgtx receptors of human neuroblastoma cells, fetal calf muscle, and chicken optic lobe but not by antibodies raised against Torpedo acetylcholine receptor, the serum of myasthenic patients, or monoclonal antibody, 35. 125I-labeled alpha Bgtx binding to the isolated receptor is blocked, with the same potency, by nicotinic agonists and antagonists, such as nicotine, neuronal bungarotoxin and, d-tubocurarine. When reconstituted in a planar lipid bilayer, the purified alpha Bgtx receptor forms cationic channels with a conductance of 50 pS. These channels are activated in a dose-dependent manner by carbamylcholine and blocked by d-tubocurarine.     

Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography.

An enzymatic activity that catalyzes ATP-dependent homologous pairing and strand exchange of duplex linear DNA and single-stranded circular DNA has been purified several thousand-fold from a human leukemic T-lymphoblast cell line. The activity was identified after chromatography of nuclear proteins on a Z-DNA column matrix. The reaction was shown to transfer the complementary single strand from a donor duplex linear substrate to a viral circular single-stranded acceptor beginning at the 5' end and proceeding in the 3' direction (5'----3'). Products of the strand-transfer reaction were characterized by electron microscopy. A 74-kDa protein was identified as the major ATP-binding peptide in active strand transferase fractions. The protein preparation described in this report binds more strongly to Z-DNA than to B-DNA.     

Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2

A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5′–3′ exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.     

Identification of tubulin from the yeast Saccharomyces cerevisiae.

A tubulin-like protein was identified in the lower eukaryote Saccharomyces cerevisiae. The following criteria were used: (i) copolymerization of the 35S-labeled yeast protein with porcine brain tubulin; (ii) immunoprecipitation of the 35S-labeled yeast protein with antiflagellar tubulin antibody; (iii) the presence of the yeast protein as a constituent of isolated yeast nuclei; and (iv) splitting of the yeast protein in a gel electrophoretic system containing sodium dodecyl sulfate that resolved the alpha- and beta-tubulin chains from other sources. This protein did not appear to have significant affinity for the plant alkaloid, Colcemid.     

Saccharomyces cerevisiae mutants that tolerate centromere plasmids at high copy number.

Two yeast (Saccharomyces cerevisiae) mutants that tolerate centromere (CEN) plasmids at high copy number have been isolated. The mutations relieve the restraint normally imposed on plasmid copy number by a cloned CEN sequence. Our CEN plasmids specify resistance to G418 and are high copy plasmids only when the mutant host cells are grown on medium containing this antibiotic. The high copy number of the plasmids is independent of the specific cloned CEN sequence and recovered plasmids show no alteration in structure or function of the CEN DNA. The efficiency with which CEN plasmids go to high copy number is increased if the mutant cell is cotransformed by another CEN plasmid. The genomic mutation responsible for the high copy number (COP) is dominant and stable, and it segregates in a Mendelian manner. Homozygous COP/COP a/alpha diploids do not tolerate CEN plasmids at high copy number, suggesting that the mutation is regulated by mating type. The genomic DNA from both mutant cells contains an altered transposon (Ty) restriction fragment that cosegregates with the COP phenotype in crosses of mutant and wild-type strains. The mutations may be transposon-mediated events that identify a gene involved in centromere or mitotic spindle function.