Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.
The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.
Stoyan
T
T
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106, USA. stoyan@lifesci.ucsb.edu
Gloeckner
G
G
Diekmann
S
S
Carbon
J
J
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002175:Candida / Q000235:genetics* / Q000378:metabolism; D002503:Centromere / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D020090:Chromosome Segregation; D002875:Chromosomes / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D000939:Epitopes; D005456:Fluorescent Dyes / Q000494:pharmacology; D005656:Fungal Proteins / Q000502:physiology*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013997:Time Factors
D000900:Anti-Bacterial Agents; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D002843:Chromatin; D004268:DNA-Binding Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D007211:Indoles; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C007293:DAPI; D004247:DNA; D008715:Methionine; D004318:Doxycycline