Telomere maintenance by the conventional DNA replication machinery and telomerase is assisted by specialized DNA helicases, nucleases and telomere binding proteins. Here, we identify the THO components at telomeres and define critical roles of this complex in telomere stability. Deletion of the THO-subunit THP2 leads to telomere shortening. We discover that telomeres contain RNA:DNA hybrid structures or R-loops which involve the long-noncoding RNA TERRA and which accumulate in thp2-D cells. Telomere length is not restored by R-loop removal upon RNase H overexpression, but by deletion of Exonuclease 1 (Exo1). Replication stress further enhances the short telomere phenotype of THP2 mutants. Similar events occur upon induced transcription of TERRA and genetic analysis links Thp2 to TERRA function. Altogether, our data indicate that THO, through the interplay with TERRA, regulates chromosome end processing activities and prevents interference with semiconservative DNA replication of telomeric DNA.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5-CGGAWATCGGATATTTTTTT-3, and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P (CDR2) -HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at alreadydescribed positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis-and trans-acting elements in C. albicans.Candida albicans is an opportunistic pathogen causing superficial to deep systemic infection in immunocompromised patients. Due to the increase in the number of people with immunodeficiencies (essentially due to AIDS, transplantation, or chemotherapy), the use of antifungal drugs is now more frequent and has led to development of drug resistance in several fungal species, especially in Candida albicans. Several resistance mechanisms have been described up to now (22). One of the more frequent is the upregulation of multidrug transporters, encoded by CDR1 and CDR2, belonging to the ATP-binding cassette transporter family (1, 2). In azole-resistant isolates, the expression of these pumps is constitutively high. The resulting drug efflux diminishes antifungal efficacy and thus protects the fungus from drug toxic effects. We demonstrated that the expression of pump-encoding genes can also be transiently upregulated by treating azole-susceptible isolates with drugs such as estradiol or fluphenazine, thus mimicking azole-resistant isolates (8). In our laboratory we are particularly interested in the regulation of the expression of multidrug transporter genes.Previous studies showed that CDR1 and CDR2 promoters contain...

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