Structure Prediction-Driven Genetics in Saccharomyces Cerevisiae Identifies an Interface between the T-RPA Proteins Stn1 and Ten1
Paschini, Margherita, Mandell, Edward K., Lundblad, Victoria, Genetics
In Saccharomyces cerevisiae, Cdc13, Stn1, and Ten1 are essential for both chromosome capping and telomere length homeostasis. These three proteins have been proposed to perform their roles at chromosome termini as a telomere-dedicated t-RPA complex, on the basis of several parallels with the conventional RPA complex. In this study, we have used several approaches to test whether a predicted a-helix in the N-terminal domain of the S. cerevisiae Stn1 protein is required for formation of the proposed t-RPA complex, in a manner analogous to the comparable helix in Rpa2. Analysis of a panel of Rpa2-OB^sup Stn1^ chimeras indicates that whether a chimeric protein contains the Rpa2 or Stn1 version of this α-helix dictates its ability to function in place of Rpa2 or Stn1, respectively. In addition, mutations introduced into a hydrophobic surface of the predicted Stn1 α-helix eliminated association with Ten1. Strikingly, allele-specific suppression of a stn1 mutation in this helix (stn1-L164D) by a ten1 mutation (ten1-D138Y) resulted in a restored Stn1-Ten1 interaction, supporting the identification of a Stn1-Ten1 interface. We conclude that Stn1 interacts with Ten1 through an α-helix, in a manner analogous to the interaction between the comparable subunits of the RPA complex.
IN the budding yeast Saccharomyces cerevisiae, a trio of essential proteins-Cdc13, Stn1, and Ten1-orchestrate a number of interactions to ensure chromosome end protection and telomere length homeostasis. In cells depleted for any of these three proteins, telomeres become subject to unregulated 59/39 resection of the C strand of telomeres, resulting in extensive singlestranded regions that signal cell cycle arrest and lethality if left unrepaired (Weinert and Hartwell 1993; Garvik et al. 1995; Lydall and Weinert 1995; Grandin et al. 1997, 2001; Vodenicharov and Wellinger 2006). The exact mechanism by which these three proteins protect chromosome termini has not been elucidated. However, there are several notable parallels between the DNA degradation that occurs at both unprotected telomeres and newly generated double-strand breaks (Ira et al. 2004; Frank et al. 2006; Vodenicharov and Wellinger 2006; Mimitou and Symington 2008; Zhu et al. 2008; Bonetti et al. 2009). In addition to an essential role in telomere capping, this complex regulates telomere length through both positive and negative mechanisms. Cdc13 interacts with the telomerase Est1 subunit, to recruit the telomerase enzyme to its site of action, thereby ensuring that telomeres do not become critically short (Nugent et al. 1996; Pennock et al. 2001; Bianchi et al. 2004). All three proteins also contribute to negative regulation of telomere length (Grandin et al. 1997, 2001; Chandra et al. 2001; Gelinas et al. 2009), although themechanism by which this second regulatory step occurs remains unclear.
Increasing evidence supports the hypothesis that Cdc13, Stn1, and Ten1 form a telomere-dedicated version of the RPA complex (Gao et al. 2007; Martin et al. 2007; Gelinas et al. 2009; Sun et al. 2009), dubbed the t-RPA complex, which acts at chromosome ends in a manner that is potentially analogous to how the canonical replication protein A (RPA) complex performs its genome-wide roles. The heterotrimeric RPA complex is the major single-strand DNA binding activity in eukaryotic cells, participating in multiple DNA transactions throughout the genome through its ability to bind singlestranded DNA with high affinity but low specificity (Wold 1997). In contrast, the proposed t-RPA complex is exclusively localized to chromosome ends as a consequence of the high specificity and affinity that the Cdc13 protein exhibits for single-stranded telomeric DNA (Nugent et al. 1996; Anderson et al. 2002). The t-RPA complex is further distinguished from the conventional RPA complex by the acquisition of an additional domain at theCterminus of Stn1,which confers a telomere-specific function (Gelinas et al. …