Rap1 in Candida albicans: an Unusual Structural Organization and a Critical Function in Suppressing Telomere Recombination

Abstract

Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.Multifunctional Rap1 (repressor activator protein 1) was first discovered in the budding yeast Saccharomyces cerevisiae as a positive transcriptional regulator of multiple growth-related genes such as the ribosomal protein genes (23). Other studies identified Rap1 as the major double-strand telomere repeat binding protein in S. cerevisiae and to be essential for maintaining telomere length and structural integrity (5, 10). Upon further analysis, Rap1 was recognized as a key component of the mating-type silencer and shown to be required for transcriptional silencing (7,43,44). These disparate observations raised fascinating questions concerning the mechanisms whereby a single protein participates in diverse functions at distinct chromosomal locations. The identification of Rap1 homologues in humans and the fission yeast resulted in yet more surprises (25,29). Both homologues were shown to be telomere-associated proteins required for proper telomere length regulation. However, instead of binding DNA directly, human and Schizosaccharomyces pombe Rap1 proteins were recruited to telomeres through interaction with other telomere proteins such as TRF2 and Taz1. Moreover, there is no evidence that the human and S. pombe proteins are involved in transcriptional regulation. Thus, although th...

Analyses of Candida Cdc13 Orthologues Revealed a Novel OB Fold Dimer Arrangement, Dimerization-Assisted DNA Binding, and Substantial Structural Differences between Cdc13 and RPA70

“…Because of the atypical translation of the CUG codon in Candida species, the CTG triplets encoding amino acids 33 and 132 of CtCdc13 were mutated to TCG to enable the expression of wild-type proteins in Escherichia coli (38). Following induction, extracts were prepared and the fusion proteins purified with Ni-nitrilotriacetic acid (NTA) chromatography as previously described (46). The fusion protein was cleaved by the ULP1 protease, and the Cdc13 fragment was purified away from the His 6 -SUMO tag by a second round of Ni-NTA affinity chromatography.…”

“…Chromatin immunoprecipitation (ChIP) of TAP-tagged Cdc13 was carried out using the same procedure as described earlier for tagged Candida Rap1 (46). Expression, purification, and crystallization of CgCdc13 OB4 .…”

“…The telomere length analysis and the twodimensional gel analysis of circular and linear telomeric DNA were performed as previously described (46).…”

Duplication and Functional Specialization of the Telomere-capping Protein Cdc13 in Candida Species

“…Telomere Analyses-The telomere length analysis and the two-dimensional gel analysis of circular and linear telomeric DNA were performed as described previously (24).…”

Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance

“…The His 6 -SUMO fusion proteins were expressed and purified essentially as previously described (52). Briefly, extracts were prepared from E. coli following IPTG (isopropyl-␤-D-thiogalactopyranoside) induction and the fusion proteins were purified using Ni-nitrilotriacetic acid (Ni-NTA) chromatography.…”