Telomere integrity is maintained through end-protection proteins that block nuclease degradation and prevent telomeres from being recognized as DNA breaks. Although less well understood, end protection proteins may also play a role in facilitating telomere replication. Here, we show that overproduction (OP) of the yeast telomere capping protein Stn1 makes cells highly sensitive to the replication inhibitors hydroxyurea (HU) and methyl-methane sulfonate (MMS). Unexpectedly, this sensitivity corresponds with Stn1 OP blocking most, if not all, aspects of the S phase checkpoint. The checkpoint kinase Rad53 is phosphorylated with normal timing in Stn1 OP cells, indicating Stn1 does not interfere with signaling steps involved in activating the checkpoint. Part of the role of Stn1 in telomere integrity is mediated through the Pol12 subunit of DNA polymerase ␣ (Pol␣). We show that overproduced Stn1 generally associates with chromosomes in HU treated and untreated cells, and, remarkably, Stn1 chromosome binding and OP checkpoint defects are rescued in pol12 mutants. We propose Stn1 normally promotes Pol␣ activity at telomeres but can be recruited through Pol12 to nontelomeric sites when overproduced. During replication stress, the mislocalized Stn1 may inappropriately promote Pol␣ in a manner that interferes with Rad53 effector mechanisms controlling replication fork integrity.

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Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric singlestranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLa complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

Plasmid engineering and molecular cloning is a virtually ubiquitous tool in biology. Although various methods have been developed for ligating DNA molecules or targeted mutagenesis of plasmids, each has its limitations. Many of the commonly used laboratory strategies are inefficient, while commercially available kits are quite costly and often specialized for highly specific circumstances. Here, we describe the SapI/AarI incision mediated plasmid editing (SIMPLE) method, which allows users to perform site-directed mutagenesis, deletions, and even short insertions into any plasmid in a single PCR reaction, using just one restriction enzyme. In addition, the SIMPLE method can be adapted to insert any sized DNA fragment into a vector using a two-step PCR approach, and can be used to ligate any number of DNA fragments with non-compatible ends in the specific order desired. The SIMPLE method provides researches an efficient and powerful tool with a broad range of applications for molecular cloning.

In the budding yeast S. cerevisiae, Stn1 is an essential protein that caps chromosome ends, and facilitates telomere replication. Previous work in our lab has shown that increasing the levels of Stn1 makes cells extremely sensitive to the replication inhibitors hydroxyurea (HU) and methyl‐methane sulfonate (MMS). Remarkably, this sensitivity corresponds with overproduced Stn1 abrogating most aspects of the S phase checkpoint, including destabilization of replication forks, de‐repression of late replication origins, and causing cells to arrest with elongated mitotic spindles. The Rad53 checkpoint kinase is activated normally in Stn1 overproducing cells, indicating Stn1 is impacting checkpoint responses downstream of the checkpoint signal transduction pathway. These observations indicate that Stn1 overproduction provides a unique reagent to probe downstream aspects of Rad53 function, and we are therefore analyzing the mechanism and genetic requirements by which Stn1 shuts down execution of the S phase checkpoint pathway. This work was funded in part by the NSF, grant number 1024792 awarded to Connie Nugent.