Positive and negative regulation of telomerase access to the telomere.

The protective caps on chromosome ends - known as telomeres - consist of DNA and associated proteins that are essential for chromosome integrity. A fundamental part of ensuring proper telomere function is maintaining adequate length of the telomeric DNA tract. Telomeric repeat sequences are synthesized by the telomerase reverse transcriptase, and, as such, telomerase is a central player in the maintenance of steady-state telomere length. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively control access of telomerase to the chromosome terminus. In yeast, positive regulation of telomerase access appears to be achieved through recruitment of the enzyme by the end-binding protein Cdc13p. In contrast, duplex-DNA-binding proteins assembled along the telomeric tract exert a feedback system that negatively modulates telomere length by limiting the action of telomerase. In mammalian cells, and perhaps also in yeast, binding of these proteins probably promotes a higher-order structure that renders the telomere inaccessible to the telomerase enzyme.

The Est3 protein is a subunit of yeast telomerase.

EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae [1] [2], as would be expected if these genes all encode components of the same complex. Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase [3] [4] [5]. In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo [1] [6] [7] [8] but are dispensable for in vitro telomerase catalytic activity [2] [9]. Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome [7] [10]. However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear. We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core. As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere. Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p. Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex.

Est1 and Cdc13 as comediators of telomerase access.

Cdc13 and Est1 are single-strand telomeric DNA binding proteins that contribute to telomere replication in the yeast Saccharomyces cerevisiae. Here it is shown that fusion of Cdc13 to the telomerase-associated Est1 protein results in greatly elongated telomeres. Fusion proteins consisting of mutant versions of Cdc13 or Est1 confer similar telomere elongation, indicating that close physical proximity can bypass telomerase-defective mutations in either protein. Fusing Cdc13 directly to the catalytic core of telomerase allows stable telomere maintenance in the absence of Est1, consistent with a role for Est1 in mediating telomerase access. Telomere length homeostasis therefore is maintained in part by restricting access of telomerase to chromosome termini, but this limiting situation can be overcome by directly tethering telomerase to the telomere.

Telomere maintenance is dependent on activities required for end repair of double-strand breaks.

Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.

Telomerase, Ku, and telomeric silencing in Saccharomyces cerevisiae.

Telomeres comprise a specialized chromosome end structure distinct from the standard nucleosomal architecture of the remainder of the genome. Telomere maintenance and chromosome stability require both replication of telomeric sequences by telomerase and telomeric end protection through binding of proteins. We have shown that Cdc13p and the heterodimer Ku are required, along with telomerase, for full telomere function, and we have proposed that Ku and Cdc13p contribute distinct roles in end protection. Ku has recently been shown to exhibit defects in transcriptional repression of telomere-proximal genes, known as telomere position effect (TPE), or telomeric silencing. We investigate here whether alterations in genes involved in the telomerase pathway also exhibit TPE defects and find that deletion or overexpression of EST1 or EST2 does not significantly affect telomeric silencing. However, telomeric silencing is derepressed upon overexpression of certain nonfunctional alleles of each. In addition, we determined that overproduction of telomerase pathway components partially alleviates the TPE defect in hdf1Delta cells. This indicates that there is genetic crosstalk between these two telomere maintenance pathways, and suggests that overproduction of telomerase pathway components may at least partially compensate for the loss of Ku in maintaining telomeric silencing.

Eat for health: a nutrition and cancer control supermarket intervention.

The growing evidence linking dietary patterns to the incidence and prevention of chronic disease has prompted a number of prominent health and scientific agencies to publish dietary guidelines for the public. Some dietary guidelines address specific diseases, such as cancer or heart disease; others focus on overall health promotion. This situation has created a demand for nutrition education and information programs for the public. Increasingly, supermarkets are seen as potential sites for effective consumer education. Eat for Health is a joint research study by the National Cancer Institute (NCI) and Giant Food Inc., a regional supermarket chain in the Washington-Baltimore area. The study's goal was to test the feasibility of supermarkets as a site for consumer nutrition education. Eat for Health's educational focus was diet and cancer control issues in the context of dietary patterns that promote health. Particular attention was paid to reduction of fat intake and increases in consumption of dietary fiber from grains, vegetables, and fruits. Analysis of program results is currently underway; data should be available in early 1990.

Strategies to improve burn center utilization.

Burn units/centers are under increasing pressure to improve resource utilization because of decreasing financial support for hospitals in general and low reimbursement for burn patients specifically. After review of occupancy and admission profiles, three strategies were implemented at the North Carolina Jaycee Burn Center to improve utilization: (1) an outreach program to attract additional burn patient referrals; (2) admission of routine plastic surgery patients to the burn center acute beds; and (3) admission of off-service ICU patients to critical care beds, depending upon availability. These strategies, especially admission of off-service ICU patients, led to an increased patient census despite an unchanged number of burn admissions and a decrease in the number of burn patient days because of earlier discharge. Policies for control of nonburn patients were essential, as were programs to educate nurses in management of other critically ill patients. These measures resulted in increased utilization and commitment of additional personnel to the burn center.