The generation of proper constitutive G-tails on yeast telomeres is dependent on the MRX complex

Abstract

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...

Replication of Telomeres and the Regulation of Telomerase

“…Cite this article as Cold Spring Harb Perspect Biol 2013;5:a010405 (Larrivee et al 2004). Furthermore, the longer G-overhangs that accumulate in yeast in late S phase are also diminished.…”

Telomerase Is Required to Protect Chromosomes with Vertebrate-type T2AG3 3′ Ends in Saccharomyces cerevisiae

“…ϩ Phleo indicates that the cells had been incubated with the radiomimetic phleomycin before analysis, and rad9⌬ indicates that the corresponding strain lacked the RAD9 gene. XhoI-digested DNA fragments were resolved on 0.75% agarose gels and the DNA was hybridized first in non-denaturing conditions with a 32 P(C 3 TA2) 3 probe to detect the vertebrate repeat-specific telomeric overhangs (A). The gel was then denatured and hybridized to the same probe to detect total vertebrate-specific telomeric DNA (B).…”

Cdc13 Telomere Capping Decreases Mec1 Association but Does Not Affect Tel1 Association with DNA Ends

“…However, the Cdc13 telomere cap may not completely inhibit the nuclease activities. Similar to DSB ends, endogenous telomeres are degraded in a Cdk1 (Cdc28)-dependent manner (Frank et al, 2006;Vodenicharov and Wellinger, 2006), and the degradation process is partially dependent on the MRX complex (Larrivee et al, 2004). DNA degradation occurs even in cells lacking the MRX complex and Exo1; for example, other nucleases such as Fen1/Rad27 are implicated in the DNA degradation (Moreau et al, 2001).…”