The telomeric 5′ end nucleotide is regulated in the budding yeast Naumovozyma castellii

Abstract

The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds-ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

Cdc13 exhibits dynamic DNA strand exchange in the presence of telomeric DNA

“…Structural and biochemical analyses have identified the OB1 and OB3 domains of Cdc13 as the ssDNA binding domains in S. cerevisiae ( 30 , 58 , 74 , 78 , 79 ). These sites are distinguished by a wide range of affinities for ssDNA, with OB1 as a low affinity binding domain for DNA ≥43 nt, and OB3 as the high affinity binding domain that can bind very short (≥8 nt) ssDNAs ( 47 , 80 ). As an isolated domain, OB3 has a 4.5-fold higher ssDNA binding affinity than full-length Cdc13, leading to the conclusion that OB3 is the DNA binding domain ( 47 , 52 ).…”

Cdc13 exhibits dynamic DNA strand exchange in the presence of telomeric DNA

“…Structural and biochemical analyses have identified the OB1 and OB3 domains of Cdc13 as the ssDNA binding domains in S. cerevisiae (10,32,41,63,65). These sites are distinguished by a wide range of affinities for ssDNA, with OB1 as a low affinity binding domain for DNA ≥ 43 nt, and OB3 as the high affinity binding domain that can bind very short (≥ 8 nt) ssDNAs (28,66). As an isolated domain, OB3 has a 4.5-fold higher ssDNA binding affinity than fulllength Cdc13, leading to the conclusion that OB3 is the DNA binding domain (28,36).…”