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...