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W D Heyer

Publications and source records attributed to W D Heyer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation.

BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.

Amino Acid Sequence↗
PubMed1996

The yeast HRS1 gene encodes a polyglutamine-rich nuclear protein required for spontaneous and hpr1-induced deletions between direct repeats.

The hrs1-1 mutation was isolated as an extragenic suppressor of the hyperrecombination phenotype of hpr1 delta cells. We have cloned, sequenced and deleted from the genome the HRS1 gene. The DNA sequence of the HRS1 gene reveals that it is identical to PGD1, a gene with no reported function, and that the Hrs1p protein contains polyglutamine stretches typically found in transcription factors. We have purified a His(6) tagged version of Hrs1p protein from E. coli and have obtained specific anti-Hrs1p polyclonal antibodies. We show that Hrs1p is a 49-kD nuclear protein, as determined by indirect immunofluorescence microscopy and Western blot analysis. The hrs1 delta null mutation reduces the frequency of deletions in wild-type and hpr1 delta backgrounds sevenfold below wild-type and rad52 levels. Furthermore, hrs1 delta cells show reduced induction of the GAL1,10 promoter relative to wild-type cells. Our results suggest that Hrs1p is required for the formation of deletions between direct repeats and that it may function in gene expression. This suggests a connection between gene expression and direct repeat recombination. In this context, we discuss the possible roles of Hrs1p and Hpr1p in initiation of direct-repeat recombination.

Amino Acid Sequence↗
PubMed1995

Use of monoclonal antibodies in the functional characterization of the Saccharomyces cerevisiae Sep1 protein.

The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also known as Xrn1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single-stranded DNA in vitro. To delineate the mechanism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity. Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1-catalyzed DNA reactions including exonuclease activity on double-stranded and single-stranded DNA, renaturation of complementary single-stranded DNA and condensation of DNA into large aggregates. All four inhibiting antibodies had no effect on the exonuclease activity of Sep1. Three antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single-stranded DNA. This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double-stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor-suppressor protein.

Antibodies, Monoclonal↗
PubMed1995

A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells lacking the SEP1 (also known as XRN1, KEM1, DST2, RAR5) gene function exhibit a number of phenotypes in cellular processes related to microtubule function. Mutant cells show increased sensitivity to the microtubule-destabilizing drug benomyl, increased chromosome loss, a karyogamy defect, impaired spindle pole body separation, and defective nuclear migration towards the bud neck. Analysis of the arrest morphology and of the survival during arrest strongly suggests a structural defect accounting for the benomyl hypersensitivity, rather than a regulatory defect in a checkpoint. Biochemical analysis of the purified Sep1 protein demonstrates its ability to promote the polymerization of procine brain and authentic S.cerevisiae tubulin into flexible microtubules in vitro. Furthermore, Sep1 co-sediments with these microtubules in sucrose cushion centrifugation. Genetic analysis of double mutant strains containing a mutation in SEP1 and in one of the genes coding for alpha- or beta-tubulin further suggests interaction between Sep1 and microtubules. Taken together these three lines of evidence constitute compelling evidence for a role of Sep1 as an accessory protein in microtubule function in the yeast S.cerevisiae.

Animals↗
PubMed1995

Identification of functional domains in the Sep1 protein (= Kem1, Xrn1), which is required for transition through meiotic prophase in Saccharomyces cerevisiae.

The Sep1 (also known as Kem1, Xrn1, Rar5, DST2/Stpbeta) protein of Saccharomyces cerevisiae is an Mr 175,000 multifunctional exonuclease with suspected roles in RNA turnover and in the microtubular cytoskeleton as well as in DNA recombination and DNA replication. The most striking phenotype of SEP1 null mutations is quantitative arrest during meiotic prophase at the pachytene stage. We have constructed a set of N- and C-terminal as well as internal deletions of the large SEP1 gene. Analysis of these deletion mutations on plasmids in a host carrying a null allele (sep1 ) revealed that at least 270 amino acids from the C-terminus of the wild-type protein were dispensable for complementing the slow growth and benomyl hypersensitivity of a null mutant. In contrast, any deletion at the N-terminus abrogated complementing activity for these phenotypes. The sequences essential for function correspond remarkably well with the regions of Sep1 that are homologous to its Schizosaccharomyces pombe counterpart Exo2. In addition, these experiments showed that, despite the high intracellular levels of Sep1, over-expression of this protein above these levels is detrimental to the cell. We discuss the potential cellular roles of the Sep1 protein as a microtubule-nucleic acid interface protein linking its suspected function in the microtubular cytoskeleton with its role as a nucleic acid binding protein.

Alleles↗
PubMed1995

Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange protein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease.

The Saccharomyces cerevisiae strand exchange protein 1 (Sep1; also referred to as Xrn1, Kem1, Rar5, or Stp beta) catalyzes the formation of hybrid DNA from model substrates in vitro. The protein is also a 5'-to-3' exonuclease active on DNA and RNA. Multiple roles for the in vivo function of Sep1, ranging from DNA recombination and cytoskeleton to RNA turnover, have been proposed. We show that Sep1 is an abundant protein in vegetative S. cerevisiae cells, present at about 80,000 molecules per diploid cell. Protein levels were not changed during the cell cycle or in response to DNA-damaging agents but increased twofold during meiosis. Cell fractionation and indirect immunofluorescence studies indicated that > 90% of Sep1 was cytoplasmic in vegetative cells, and indirect immunofluorescence indicated a cytoplasmic localization in meiotic cells as well. The localization supports the proposal that Sep1 has a role in cytoplasmic RNA metabolism. Anti-Sep1 monoclonal antibodies detected cross-reacting antigens in the fission yeast Schizosccharomyces pombe, in Drosophila melanogaster embryos, in Xenopus laevis, and in a mouse pre-B-cell line.

Animals↗
PubMed1994

A multifunctional exonuclease from vegetative Schizosaccharomyces pombe cells exhibiting in vitro strand exchange activity.

A 140-kDa polypeptide (p140) has been purified over 2000-fold from vegetative Schizosaccharomyces pombe cells using an assay of homologous pairing and strand exchange between linear double-stranded DNA (dsDNA) and circular single-stranded DNA (ssDNA) in vitro. Electron microscopic analysis of the reaction products showed displacement of one strand of the linear duplex DNA by the circular ssDNA molecule. In addition, the protein contained 5' to 3' exonuclease activity on ssDNA and dsDNA (with a 50-fold preference on the single-stranded substrate) as well as on single-stranded RNA. Furthermore, p140 was capable of renaturing complementary ssDNA as shown by S1 nuclease assays. p140 behaved like a monomer in solution under reaction conditions. Direct comparison of the biochemical properties, sequence analysis, and cross-reactivity to a monoclonal antibody suggests that p140 is probably identical with ExoII, purified from S. pombe meiotic cells as a ssDNA exonuclease (Szankasi, P., and Smith, G. R. (1992) Biochemistry 31, 6769-6773). Given the diverse activities of p140, the protein might be involved in DNA and/or RNA metabolism in vivo.

Amino Acid Sequence↗
PubMed1994

Schizosaccharomyces pombe fatty acid synthase mediates DNA strand exchange in vitro.

During purification of a strand exchange activity from Schizosaccharomyces pombe using the three-strand reaction of double-stranded linear and circular single-stranded DNA, we identified p190/210 as an activity that stimulated the strand exchange activity of p140exo2 by about 10-fold. The accompanying report (Käslin, E., and Heyer, W.-D. (1994) J. Biol. Chem. 269, 0000-0000) described the purification and characterization of p140exo2, likely to be the S. pombe homolog of the Saccharomyces cerevisiae strand exchange protein p175SEP1. Here, we report the purification of p190/210 from S. pombe cells and its identification as fatty acid synthase (FAS). S. pombe FAS (p190/210) binds to single-stranded and double-stranded DNA, leading to condensation of DNA into large aggregates. In addition, it is capable of renaturing complementary single-stranded DNA. Besides stimulating the strand exchange activity of p140exo2, FAS (p190/210) itself exhibits strand exchange activity provided the double-stranded substrate has single-stranded tails. We propose a probable mechanism for the action of FAS (p190/210) during DNA strand exchange in vitro. Since FAS (p190/210) is highly unlikely to have a role in homologous recombination in vivo, we discuss the implications of our data on the interpretation of other homologous pairing and strand exchange proteins purified from eukaryotes using this or similar assays.

Amino Acid Sequence↗
PubMed1994

The search for the right partner: homologous pairing and DNA strand exchange proteins in eukaryotes.

Finding the right partner is a central problem in homologous recombination. Common to all models for general recombination is a homologous pairing and DNA strand exchange step. In prokaryotes this process has mainly been studied with the RecA protein of Escherichia coli. Two approaches have been used to find homologous pairing and DNA strand exchange proteins in eukaryotes. A biochemical approach has resulted in numerous proteins from various organisms. Almost all of these proteins are biochemically fundamentally different from RecA. The in vivo role of these proteins is largely not understood. A molecular-genetical approach has identified structural homologs to the E. coli RecA protein in the yeast Saccharomyces cerevisiae and subsequently in other organisms including other fungi, mammals, birds, and plants. The biochemistry of the eukaryotic RecA homologs is largely unsolved. For the fungal RecA homologs (S. cerevisiae RAD51, RAD55, RAD57, DMC1; Schizosaccharomyces pombe rad51; Neurospora crassa mei3) a role in homologous recombination and recombinational repair is evident. Besides recombination, homologous pairing proteins might be involved in other cellular processes like chromosome pairing or gene inactivation.

Adenosine Triphosphate↗
PubMed1994

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175SEP1 arrest at pachytene during meiotic prophase.

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175SEP1, enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.

Chromosomes, Fungal↗
PubMed1993

Identification of a chicken RAD52 homologue suggests conservation of the RAD52 recombination pathway throughout the evolution of higher eukaryotes.

Degenerate oligonucleotides encoding conserved regions of the Rad52 protein of S. cerevisiae and its homologue, the Rad22 protein of S. pombe, were used to clone a chicken RAD52 counterpart by the polymerase chain reaction. Sequence comparison of the chicken and yeast proteins reveals a strongly conserved region between positions 40 and 178 of the chicken Rad52 sequence indicating that this part of the protein is under strong evolutionary pressure. The first 39 amino acids and the 3' end of the chicken Rad52 homologue does not share significant similarity with the yeast proteins. High abundance of the mRNA in testis makes it likely that the chicken Rad52 protein plays a role in meiotic recombination.

Amino Acid Sequence↗
PubMed1991

Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication.

Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.

Amino Acid Sequence↗
PubMed1990

An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A.

Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes. An SSB was previously purified from the yeast Saccharomyces cerevisiae. This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination. We have cloned and functionally analyzed the gene encoding this protein. DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide. Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro. Therefore, we named the S.cerevisiae gene RPA1. RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth. Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of a protein from Saccharomyces cerevisiae that binds tightly to single-stranded DNA and stimulates a cognate strand exchange protein.

A single-stranded DNA binding protein (yeast SSB protein) was purified to near-homogeneity from mitotic Saccharomyces cerevisiae cells. The Mr 34,000 protein specifically eluted at high salt (approximately 1200 mM NaCl) during chromatography on a single-stranded DNA-cellulose column. The protein formed stable complexes with single-stranded DNA in an apparent cooperative fashion. As judged from titration and competition experiments, the affinity of the protein was much higher for single-stranded DNA than for double-stranded DNA or single-stranded RNA. The SSB protein also was found to stimulate the strand exchange reaction between linear M13mp19 RF DNA and circular M13mp19 viral DNA as catalyzed by a yeast strand exchange protein previously purified in this laboratory [Kolodner, R., Evans, D. H., & Morrison, P. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5660-5664]. Titration experiments showed maximum stimulation of joint molecule formation at a stoichiometry of about 1 Mr 34,000 monomer yeast SSB per 18 nucleotides of single-stranded DNA. Kinetic experiments demonstrated at least an 18-fold increase in the rate of strand exchange due to the presence of the SSB in reactions where the amount of strand exchange protein was limiting. The yeast SSB protein stimulated the Escherichia coli RecA protein in the strand exchange reaction involving linear M13mp19 RF DNA and circular M13mp19 viral DNA as efficiently as E. coli SSB. However, the E. coli SSB protein did not substitute for the yeast SSB protein in reactions with the yeast strand exchange protein. This suggests that the stimulation of the yeast strand exchange protein by the yeast SSB may involve specific protein/protein interactions.

Chromatography, Affinity↗
PubMed1988

DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination host spot allele ade6-M26.

The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe. It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis. A DNA fragment of 3043 nucleotides has been sequenced. It complements ade6 mutations when present on plasmids. An uninterrupted open reading frame of 552 amino acid residues was identified. A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed. Twelve ade6 mutant alleles have been isolated. The sequence alterations of four mutant alleles have been determined. Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation. Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons. They are suppressed by defined tRNA nonsense suppressors of the UGA type. The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations. Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.

Alleles↗
PubMed1988

Renaturation of DNA by a Saccharomyces cerevisiae protein that catalyzes homologous pairing and strand exchange.

A protein from mitotic Saccharomyces cerevisiae cells that catalyzes homologous pairing and strand exchange was analyzed for the ability to catalyze other related reactions. The protein was capable of renaturing complementary single-stranded DNA as evidenced by S1 nuclease assays and analysis of the reaction products by agarose gel electrophoresis and electron microscopy. Incubation of the yeast protein with complementary single-stranded DNA resulted in the rapid formation of large aggregates which did not enter agarose gels. These aggregates contained many branched structures consisting of both single-stranded and double-stranded DNA. These reactions required stoichiometric amounts of protein but showed no ATP requirement. The protein formed stable complexes with both single-stranded and double-stranded DNA, showing a higher affinity for single-stranded DNA. The binding to single-stranded DNA resulted in the formation of large protein:DNA aggregates. These aggregates were also formed in strand-exchange reactions and contained both substrate and product DNAs. These results demonstrate that the S. cerevisiae strand-exchange protein shares additional properties with the Escherichia coli recA protein which, by analogy, gives further indication that it might be implicated in homologous recombination.

DNA, Single-Stranded↗