Figure 1.

Identification of a hypomophic mutation in erg11-1 that dramatically sensitizes S. pombe to chronic treatment with HU. (A) The newly screened hus41 mutant was rescued by erg11. Standard spot assays were used to assess the HU sensitivity of wild type, rad3, and hus41 carrying an empty vector (V), and hus41 carrying the same vector expressing erg11 or mutant erg11-1 cloned from hus41. The 2 × 10⁷ cells/ml of logarithmically growing S. pombe were diluted in fivefold steps and spotted onto YE6S plates as the control or YE6S plates containing 5.0 mM HU. The plates were incubated at 30° for 3 days before they were photographed. The dye Phloxin B was added to the medium in this experiment as the indicator of cell lethality. (B) DNA sequencing of the erg11 cloned from hus41 identified a single G-to-A mutation that causes G189D amino acid change in the enzyme. (C) Overexpression of mutant Erg11 in cells lacking the erg11 gene generated minimal HU sensitivity as determined by standard spot assay. (D) Integration of mutant erg11-1 in the erg11 genomic locus sensitized S. pombe to HU to the same level as the hus41 mutant. The erg11-1 mutation was integrated at the genomic locus as illustrated in Figure S3. As a control, wild-type erg11 was integrated by the same method. The HU sensitivity of the resulting integrants was assessed by spot assay and compared with that of wild-type, rad3, cds1, chk1, and hus41 cells. (E) The mutated glycine residue in Erg11 is conserved from yeasts to humans. Regional amino acid sequences of human CYP51, S. cerevisae, and S. pombe Erg11 were aligned together using the numbering of the amino acids in human CYP51 (numbers on top). Number 218 is in boldface type to indicate the mutated glycine residue in hus41. The highly conserved two histidines and four charged residues surrounding the mutated glycine residue are also numbered. (F) The mutated residue is located outside the catalytic center. The crystal structure of human CYP51 is shown by POLYVIEW (Porollo et al. 2004). The N and C termini are marked by N and C, respectively. Also shown are cyclo-hepta-amylose co-crystalized with the N terminus and the inhibitor econazole that occupies the catalytic center (Strushkevich et al. 2010). The mutated region is enlarged to show the surrounding four charged residues and the two histidines numbered in E. (G) Overexpression of human CYP51 and S. cerevisiae Erg11 in S. pombe partially rescued the erg11-1 mutant. HU sensitivity of wild-type or erg11-1 mutant cells carrying an empty vector or the same vector expressing human Cyp51 or S. cerevisiae Erg11 was assessed by spot assay. Expression of the human Cyp51 and S. cerevisiae Erg11 was under the control of nmt1 promoter.