C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

Abstract

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(Gl13T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele ofRAP], rapl-S. These telomere deficiency (rapl') alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rapl-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the R4D52 or RADI gene product. Third, chromosome loss and nondisjunction rates are eleva ted 15-to 30-fold above wild-type levels. Sequencing analysis has shown that each rapl' allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.Telomeres, the structures present at the ends of linear eukaryotic chromosomes, are essential for the stability and complete replication of the chromosome. Telomeric DNA, at its extreme terminus, is composed of a variable number of simple sequence repeats. These repeats usually contain a G-rich strand, oriented in a 5'-to-3' direction toward the terminus. In contrast to other genomic sequences, telomeres are replicated inexactly. The size and, in some organisms, the sequence of an individual telomere vary among different cells of a population (60). The average size of telomeric DNA appears to be maintained through a regulated equilibrium between the loss and gain of telomeric sequences (36,45,46,60). One component involved in this process is the ribonucleoprotein complex telomerase (16-18, 37, 48, 49, 59). This enzyme, identified in both ciliate and mammalian cells, is capable of catalyzing the addition of the G-rich simple sequences onto the 3' end of single-stranded substrates, utilizing a sequence within its RNA component as a template. A similar activity is likely to explain the properties of telomere addition in yeasts and other organisms in which telomerase has not yet been identified (60). The essentiality of maintaining telomeric sequences is underscored by the recombinational instability, chromosome loss, and lethality caused by loss of part or all of telomeric DNA (34, 60).Telomeres are packaged in vivo into unique nonnucleosomal complexes, f...

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

“…Thus, the telomere growth observed after POT1a depletion occurs in nondividing cells and hence is different from the progressive elongation that occurs over many cell divisions when Tetrahymena cells are kept in continuous culture (30). It also differs from the telomere elongation observed in various yeast mutants (28,34) and mammalian cells expressing mutant forms of POT1 (7,32), as in these situations the elongation also occurs in dividing cells.…”

“…Thus, while the participation of POT1, or POT1 homologs, in processes such as telomere length regulation or C-strand processing may vary between organisms, it appears that the essential function of POT1 is to prevent a catastrophic telomeric DNA damage response and that this function has been conserved in widely diverse organisms. The telomere length phenotype caused by depletion of POT1a is unusual because unlike most defects in telomere length regulation where the increase or decrease in length occurs gradually over many cell divisions (7,28,32,34), the telomere growth in POT1a-deficient cells happens after the cells have stopped dividing. Moreover, the extent of growth can be quite dramatic, with some rDNA telomeres reaching in excess of 10 kb or Ͼ30-fold their normal length.…”

Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation

“…We also think that pol12-216 stn1-13 lethality is unlikely to be caused by a telomerase regulation defect. Neither single mutant shows particularly elevated telomere length under the conditions of the experiment (23°C), and the apparently rapid lethality we observe is inconsistent with known effects of unregulated telomerase action in yeast (Kyrion et al 1992).…”

“…Maintenance of telomere length about a fixed average value is achieved through a mechanism that appears to measure not the TG-repeat tract length per se, but, rather, the number of Rap1 proteins bound to it (Marcand et al 1997;Ray and Runge 1999a). The C terminus of Rap1 negatively regulates telomere elongation in cis (Kyrion et al 1992;Marcand et al 1996), a function that requires two additional proteins, Rif1 and Rif2, both of which interact physically with this domain of Rap1 (Hardy et al 1992;Moretti et al 1994;Wotton and Shore 1997). The inhibition of telomerase addition by Rap1-Rif1/2 complexes increases linearly as a function of telomere tract length (and presumably the number of Rap1-Rif1/2 complexes bound) through a mechanism that is at present unknown.…”

New ways not to make ends meet: telomerase, DNA damage proteins and heterochromatin

“…Position e ect variegation is characterized by epigenetic heterochromatin states that are semi-stable for several cell divisions, with a gene under its in¯uence switching between silenced or non-silenced states. Telomeric silencing in yeast shares several molecular requirements with silencing of the chromosome-internal mating type loci (Aparicio et al, 1991), including the recruitment of Sir3p and Sir4p proteins by the C-terminal domain of Rap1p (Kyrion et al, 1992). This suggested a molecular mechanistic link between the telomeric structures underlying telomere capping/uncapping status and the two functional states ± repressed or active ± that characterize heterochromatin.…”