Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.     

DNA-dependent in vitro synthesis of ribosomal proteins,protein elongation factors,and RNA polymerase subunit α: Inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) had been implicated in stringent control, we examied the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage λfus3 and λrif^d18. λfus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit α, in addition to genes for approximately 27 r proteins. λrif^d18 carries genes for EF-Tu, RNA polymerase subunits β and β^I, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2–0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit α, as well as rRNAs.