Victoria Lundblad

Cdc13p: A Single-Strand Telomeric DNA-Binding Protein with a Dual Role in Yeast Telomere Maintenance

The CDC13 gene has previously been implicated in the maintenance of telomere integrity in Saccharomyces cerevisiae. With the use of two classes of mutations, here it is shown that CDC13 has two discrete roles at the telomere. The cdc13-2est mutation perturbs a function required in vivo for telomerase regulation but not in vitro for enzyme activity, whereas cdc13-1ts defines a separate essential role at the telomere. In vitro, purified Cdc13p binds to single-strand yeast telomeric DNA. Therefore, Cdc13p is a telomere-binding protein required to protect the telomere and mediate access of telomerase to the chromosomal terminus.

Cdc13 delivers separate complexes to the telomere for end protection and replication.

In Saccharomyces cerevisiae, the telomere binding protein Cdc13 mediates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection. We show here that delivery of the Stn1 protein to the telomere, by fusing the DNA binding domain of Cdc13 (DBD(CDC13)) to Stn1, is sufficient to rescue the lethality of a cdc13 null strain and, hence, provide end protection. Telomere replication is still defective in this strain, but can be restored by delivering telomerase to the telomere as a DBD(CDC13)-telomerase fusion. These results establish Stn1 as the primary effector of chromosome end protection, whereas the principal function of Cdc13 is to provide a loading platform to recruit complexes that provide end protection and telomere replication.

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.

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.

Defects in mismatch repair promote telomerase-independent proliferation

Mismatch repair has a central role in maintaining genomic stability by repairing DNA replication errors and inhibiting recombination between non-identical (homeologous) sequences. Defects in mismatch repair have been linked to certain human cancers, including hereditary non-polyposis colorectal cancer (HNPCC) and sporadic tumours. A crucial requirement for tumour cell proliferation is the maintenance of telomere length, and most tumours achieve this by reactivating telomerase. In both yeast and human cells, however, telomerase-independent telomere maintenance can occur as a result of recombination-dependent exchanges between often imperfectly matched telomeric sequences. Here we show that loss of mismatch-repair function promotes cellular proliferation in the absence of telomerase. Defects in mismatch repair, including mutations that correspond to the same amino-acid changes recovered from HNPCC tumours, enhance telomerase-independent survival in both Saccharomyces cerevisiae and a related budding yeast with a degree of telomere sequence homology that is similar to human telomeres. These results indicate that enhanced telomeric recombination in human cells with mismatch-repair defects may contribute to cell immortalization and hence tumorigenesis.

Introduction of DNA into Yeast Cells

The most commonly used yeast transformation protocol is the lithium acetate procedure (described here). It is reasonably fast and provides a transformation efficiency of 105 to 106 transformants/μg. This efficiency rivals that achieved for most, but not all, strains with the more difficult and time‐consuming spheroplast procedure presented here. However, the fastest and easiest of the transformation methods is electroporation, as described in this unit. For a number of strains, electroporation offers the highest transformation efficiency, and may prove especially useful with limiting quantities of transforming DNA. Unlike the lithium acetate procedure, however, electroporation saturates at low DNA levels, restricting its general utility.

The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini.

ABSTRACT The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80tel alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Kus role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80tel alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

Telomeres and Telomerase: A Simple Picture Becomes Complex

The results presented at this meeting indicated that there are many more levels of regulation of telomerase activity and telomere length than initially suspected. Analysis of the mouse and human telomerase RNA components has already shown developmental and tissue-specific regulation, as well as increased expression during tumor proliferation. As the genes encoding both the hypothesized telomerase inhibitor and protein subunits of the human enzyme are cloned, a detailed molecular dissection of the pattern of expression of these additional components will become possible, revealing their potential contributions to telomerase regulation. An additional critical experiment will be a direct in vivo test of the hypothesis that telomerase reactivation is an obligatory step in oncogenesis, via genetic manipulations to ask first, if tumor progression is impaired when telomerase genes are inactivated and second, if tumor formation can be promoted when telomerase expression is turned on in normal tissues. Crucial to the understanding of these experimental alterations in telomerase expression will be a more detailed understanding of the potential contribution of alternative telomerase-independent pathways. Finally, from the standpoint of the basic scientist, the stage is also set for a series of key experiments, including reconstitution of the enzyme from purified components and a molecular investigation of how telomerase interacts with components of the telosome.In spite of the complexities of telomerase regulation, the clinical reports presented at this meeting continued to support the initial enthusiasm for the diagnostic, prognostic, and therapeutic uses of telomerase assays and potential telomerase inhibitors. The original all-or-none assessment of telomerase activity in cancer versus normal cells has proven to be an oversimplification, indicating that an increasingly sophisticated analysis of telomerase levels will be required for meaningful clinical interpretations. However, the clinical studies continue to show both the value of telomerase as a tumor marker and the ways in which knowledge about telomerase activity can be used to aid clinicians in their care of cancer patients.

DNA ends: maintenance of chromosome termini versus repair of double strand breaks.

This review focuses on the factors that define the differences between the two types of DNA ends encountered by eukaryotic cells: telomeres and double strand breaks (DSBs). Although these two types of DNA termini are functionally distinct, recent studies have shown that a number of proteins is shared at telomeres and sites of DSB repair. The significance of these common components is discussed, as well as the types of DNA repair events that can compensate for a defective telomere.

Yeast Vectors and Assays for Expression of Cloned Genes

This unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed.