Telomerase recruitment in Saccharomyces cerevisiae is not dependent on Tel1-mediated phosphorylation of Cdc13.

In Saccharomyces cerevisiae, association between the Est1 telomerase subunit and the telomere-binding protein Cdc13 is essential for telomerase to be recruited to its site of action. A current model proposes that Tel1 binding to telomeres marks them for elongation, as the result of phosphorylation of a proposed S/TQ cluster in the telomerase recruitment domain of Cdc13. However, three observations presented here argue against one key aspect of this model. First, the pattern of Cdc13 phosphatase-sensitive isoforms is not altered by loss of Tel1 function or by mutations introduced into two conserved serines (S249 and S255) in the Cdc13 recruitment domain. Second, an interaction between Cdc13 and Est1, as monitored by a two-hybrid assay, is dependent on S255 but Tel1-independent. Finally, a derivative of Cdc13, cdc13-(S/TQ)11→(S/TA)11, in which every potential consensus phosphorylation site for Tel1 has been eliminated, confers nearly wild-type telomere length. These results are inconsistent with a model in which the Cdc13-Est1 interaction is regulated by Tel1-mediated phosphorylation of the Cdc13 telomerase recruitment domain. We propose an alternative model for the role of Tel1 in telomere homeostasis, which is based on the assumption that Tel1 performs the same molecular task at double-strand breaks (DSBs) and chromosome termini.

RPA-like proteins mediate yeast telomere function.

Cdc13, Stn1 and Ten1 are essential yeast proteins that both protect chromosome termini from unregulated resection and regulate telomere length. Cdc13, which localizes to telomeres through high-affinity binding to telomeric single-stranded DNA, has been extensively characterized, whereas the contribution(s) of the Cdc13-associated Stn1 and Ten1 proteins to telomere function have remained unclear. We show here that Stn1 and Ten1 are DNA-binding proteins with specificity for telomeric DNA substrates. Furthermore, Stn1 and Ten1 show similarities to Rpa2 and Rpa3, subunits of the heterotrimeric replication protein A (RPA) complex, which is the major single-stranded DNA-binding activity in eukaryotic cells. We propose that Cdc13, Stn1 and Ten1 function as a telomere-specific RPA-like complex. Identification of an RPA-like complex that is targeted to a specific region of the genome suggests that multiple RPA-like complexes have evolved, each making individual contributions to genomic stability.

DNA damage response and mutagenesis in mouse embryonic stem cells.

Mutation in embryonic stem (ES) cells can potentially compromise multiple cell lineages and affect the well-being of subsequent generations. Thus, ES cells require sensitive mechanisms to maintain genomic integrity. One mechanism involves suppression of mutation. A complementary mechanism is to regulate the cell cycle checkpoint and facilitate cell death. Here, we describe the detailed protocols we have used to investigate DNA damage response and mutagenesis in mouse ES cells.

Homology among telomeric end-protection proteins.

Telomere maintenance and end protection are essential for the survival and proliferation of eukaryotic cells, leading to the prediction that components of this system would be highly conserved. In practice, however, evidence for homology among these factors has been elusive, and, in the case of the known end-protection proteins, evolutionary relationships have been postulated largely on the basis of protein structural and functional similarity alone. Here we report support from sequence profile analyses for a significant and specific evolutionary relationship among OB-fold telomeric end-protection factors.

Mechanisms of chromosome-end protection.

Telomeres must protect chromosome ends from being recognized and processed as double-strand breaks. Identification of the factors involved in end protection, and the mechanisms by which they "cap" chromosome termini, is crucial in understanding how the cell distinguishes between a double-strand break and a normal telomere end. Recent work has characterized the similarities and potential differences between the pathways utilized by multiple organisms in maintaining telomere ends. One unifying concept that has clearly emerged is that chromosome-end protection is necessary in maintaining genetic stability and preventing oncogenesis.

Embryonic stem cells and somatic cells differ in mutation frequency and type.

Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss/reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.