Rudimentary G-quadruplex-based telomere capping in Saccharomyces cerevisiae.

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3' overhang inhibits 5'→3' resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo.

Cdc13 N-terminal dimerization, DNA binding, and telomere length regulation.

The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

Isolation of G-quadruplex DNA using NMM-sepharose affinity chromatography.

DNA can adopt a variety of non-standard conformations, including structures known as G-quadruplexes (G4-DNA), which consist of stacked tetrads of guanines. There are growing indications that G4-DNA is of biological importance, including evidence that it plays roles in telomere function, DNA recombination and the regulation of transcription and translation. However, it has been difficult to obtain direct, physical evidence for the presence of G-quadruplex DNA in vivo due, in part, to a lack of tools for G4-DNA identification. Here, we describe a method for coupling the G4-DNA binding ligand N-methyl mesoporphyrin IX (NMM) to a Sepharose resin, and demonstrate the ability of the resin to bind tightly and selectively to DNA oligonucleotides with the capacity to form G4-DNA. This technique might also be extended to examine genomic distributions of G4-DNA isolated from in vivo sources.

Significant correlation of species longevity with DNA double strand break recognition but not with telomere length.

The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSBs) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed.

Functional unresponsiveness and replicative senescence of myeloid leukemia antigen-specific CD8+ T cells after allogeneic stem cell transplantation.

PURPOSE: The therapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) for patients with myeloid malignancies has been attributed in part to a graft-versus-leukemia effect that is dependent on donor T lymphocytes. CD8(+) T-cell responses to MHC class I-restricted tumor epitopes, not just allogeneic antigens, may help mediate antileukemia effects after HSCT, but the specificity and function of such cells are not completely understood. EXPERIMENTAL DESIGN: We examined the diversity, phenotype, and functional potential of leukemia-associated antigen-specific CD8(+) T cells in patients with myeloid leukemia following allogeneic HSCT. Screening for antigen-specific T cells was accomplished with a peptide/MHC tetramer library. RESULTS: Patients with acute myelogenous leukemia or chronic myelogenous leukemia in remission following HSCT exhibited significant numbers of peripheral blood CD8(+) T cells that recognized varying combinations of epitopes derived from leukemia-associated antigens. However, these cells failed to proliferate, release cytokines, or degranulate in response to antigen-specific stimuli. As early as 2 months after HSCT, CD8(+) T cells from patients were predominantly CD28(-) CD57(+) and had relatively short telomeres, consistent with cellular senescence. CONCLUSIONS: Circulating leukemia-specific CD8(+) T cells are prominent in myeloid leukemia patients after HSCT, but such cells are largely functionally unresponsive, most likely due to replicative senescence. These findings carry important implications for the understanding of the graft-versus-leukemia effect and for the rational design of immunotherapeutic strategies for patients with myeloid leukemias.

In vivo veritas: using yeast to probe the biological functions of G-quadruplexes.

Certain guanine-rich sequences are capable of forming higher order structures known as G-quadruplexes. Moreover, particular genomic regions in a number of highly divergent organisms are enriched for such sequences, raising the possibility that G-quadruplexes form in vivo and affect cellular processes. While G-quadruplexes have been rigorously studied in vitro, whether these structures actually form in vivo and what their roles might be in the context of the cell have remained largely unanswered questions. Recent studies suggest that G-quadruplexes participate in the regulation of such varied processes as telomere maintenance, transcriptional regulation and ribosome biogenesis. Here we review studies aimed at elucidating the in vivo functions of quadruplex structures, with a particular focus on findings in yeast. In addition, we discuss the utility of yeast model systems in the study of the cellular roles of G-quadruplexes.