During telomere replication in yeast, chromosome ends acquire an S-phase-specific overhang of the guanosine-rich strand. Here it is shown that in cells lacking Ku, a heterodimeric protein involved in nonhomologous DNA end joining, these overhangs are present throughout the cell cycle. In vivo cross-linking experiments demonstrated that Ku is bound to telomeric DNA. These results show that Ku plays a direct role in establishing a normal DNA end structure on yeast chromosomes, conceivably by functioning as a terminus-binding factor. Because Ku-mediated DNA end joining involving telomeres would result in chromosome instability, our data also suggest that Ku has a distinct function when bound to telomeres.
The precise DNA arrangement at chromosomal ends and the proteins involved in its maintenance are of crucial importance for genome stability. For the yeast Saccharomyces cerevisiae, this constitutive DNA configuration has remained unknown. We demonstrate here that Gtails of 12-14 bases are present outside of S phase on normal yeast telomeres. Furthermore, the Mre11p protein is essential for the proper establishment of this constitutive end-structure. However, the timing of extended G-tails occurring during S phase is not affected in strains lacking Mre11p. Thus, G-tails are present on yeast chromosomes throughout the cell cycle and the MRX complex is required for their normal establishment. The physical ends of eukaryotic chromosomes, the telomeres, have a very conserved structure and are essential for genome stability (for review, see Blackburn 2001;Chakhparonian and Wellinger 2003). Short, direct DNA repeats constitute the underlying telomeric DNA and the strand running 5Ј to 3Ј toward the end of the chromosomes is usually rich in guanines (the G-rich strand). Lagging-strand synthesis always occurs on this G-rich strand and will leave a short gap at the 5Ј end of the newly synthesized C-rich strand. This gap cannot be filled in by repair, and a 3Ј G-rich overhang, called G-tail, remains. On the other end, leading-strand synthesis is thought to produce a blunt extremity. However, studies of the terminal DNA arrangement in a variety of organisms suggest that a G-tail is a conserved motif for all telomeres (Chakhparonian and Wellinger 2003). Thus, the question arises as to how the blunt-ended DNA ends generated by leading-strand synthesis are converted into ends with a G-tail.Studies in the yeast Saccharomyces cerevisiae have shown that its telomeres acquire detectable G-tails late in S phase, after conventional replication (Wellinger et al. 1993a,b). Moreover, at least on the ends of a linear plasmid, G-tails occur on both, leading-and laggingstrand ends . Surprisingly, these S-phase-specific G-tails can also be detected in cells lacking telomerase, the main activity responsible for replicating telomeric G-strands (Dionne and Wellinger 1996). Collectively, these results suggest that the blunt end left after completion of leading-strand synthesis is processed into an end with a G-tail, presumably by nuclease/helicase activities . Analyses of the requirements to establish a normal telomeric DNA endstructure are hampered by the fact that for wild-type yeast cells, the precise DNA arrangement outside of S phase is unknown.Recent studies on the Mre11p/Rad50p/Xrs2p (MRX) proteins, an evolutionarily conserved complex involved in a number of processes in mitosis and meiosis, revealed that this complex may play a key role in telomere length maintenance in humans, plants, and yeasts (for review, see Haber 1998; D'Amours and Jackson 2002). Yeast cells harboring a deletion of any one of these genes are viable, but display shortened telomeric repeat tracts (Kironmai and Muniyappa 1997; Boulton and Jackson 1998). The Mre11p p...
Maintaining telomeric DNA at chromosome ends is essential for genome stability. In virtually all organisms the telomerase enzyme provides this function; however, telomerase-independent mechanisms also exist. These latter mechanisms rely on recombination pathways to replenish telomeric DNA and extrachromosomal DNA may also be implicated. Here, we report that in Saccharomyces cerevisiae cells, extrachromosomal circular DNA occurs for both subtypes of telomerase-independent telomere-maintenance mechanisms. This DNA consists of circular molecules of full-length subtelomeric repeat elements in type I cells, and very heterogeneously sized circles of telomeric repeat DNA in type II cells that are at least partially single stranded. Surprisingly, both type I and type II cells can adapt to a loss of the normally essential telomere-capping protein Cdc13p by inducing an alternate and reversible state of chromosome ends. Chromosome capping, therefore, is not strictly dependent on canonical capping proteins, such as Cdc13p, but can be achieved by alternate mechanisms.
The Yku heterodimer from Saccharomyces cerevisiae, comprising Yku70p and Yku80p, is involved in the maintenance of a normal telomeric DNA end structure and is an essential component of nonhomologous end joining (NHEJ). To investigate the role of the Yku70p subunit in these two different pathways, we generated C-terminal deletions of the Yku70 protein and examined their ability to complement the phenotypes of a yku70 ؊ strain. Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA binding activity and causes a yku ؊ phenotype; telomeres are shortened, and NHEJ is impaired. Using conditions in which at least as much mutant protein as full-length protein is normally detectable in cell extracts, deleting only 25 C-terminal amino acids of Yku70p results in no measurable effect on DNA binding of the Yku protein, and the cells are fully proficient for NHEJ. Nevertheless, these cells display considerably shortened telomeres, and significant amounts of singlestranded overhangs of the telomeric guanosine-rich strands are observed. Co-overexpression of this protein with Yku80p could rescue some but not all of the telomere-related phenotypes. Therefore, the C-terminal domain in Yku70p defines at least one domain that is especially involved in telomere maintenance but not in NHEJ.
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