The maintenance of telomeres in the budding yeast Naumovozyma castellii

Sammanfattning: Linear chromosomes of eukaryotic cells require the presence of functional nucleoprotein terminal structures, known as telomeres, to protect the integrity of the genome. The telomere is a highly dynamic and regulated structure constituted by short tandem DNA repeats rich in guanine nucleotides that extent as double-stranded DNA ending in a single-stranded 3′ overhang. An abundant number of proteins bind these sequences, like the double-stranded binding protein Rap1 and the single-stranded binding protein Cdc13. Telomeres protect the genomic DNA from end-to-end fusions, degradation and recognition as damaged DNA by the DNA repair machinery of the cell.Many factors contribute to the progressive shortening of telomeres during each replication cycle, including the inability of the canonical DNA replication machinery to fully replicate the telomere, a phenomenon known as the end replication problem. The enzyme telomerase, a DNA polymerase with reverse transcriptase activity, prevents progressive shortening by adding telomeric repeats to the single stranded end of the chromosome using its internal RNA molecule as template. Eukaryotes solve the end replication problem with the use of telomerase but, in its absence, some cells develop telomerase-independent mechanisms for telomere maintenance, like the recombination based alternative lengthening of telomeres (ALT) mechanism that has been observed in both yeast cells and human tumors.My doctoral thesis focuses on studying the maintenance of telomeres in the budding yeast Naumovozyma castellii. I approached the studies from two perspectives: the structural maintenance of the telomeres and the telomerase-independent telomere maintenance. I investigated how Rap1 and Cdc13 provide protection to the 3′ overhang from exonuclease degradation in vitro and discovered a previously undescribed function of Rap1: the ability to protect short telomeric overhangs. I investigated the double-stranded and single-stranded junction of the telomeres and determined, for the first time in yeast, that the terminal 5' end nucleotide is regulated to contain primarily an adenine nucleotide in N. castellii. With knowledge of the DNA structure and with the implementation of our protection assays, I proposed a model that describes how the binding of Rap1 and Cdc13 protects the telomere from enzymatic degradation. To investigate the genetic requirements for the establishment of the ALT mechanism I first characterized the RAD52 gene, coding for the main homologous recombination gene in yeast. To investigate if the ALT mechanism of N. castellii relies on homologous recombination I designed multiple strains and evaluated their rate of senescence and telomere structure. I found that the establishment of the efficient ALT mechanism of N. castellii requires homologous recombination mediated by RAD52 and RAD51 gene function. These findings expand the understanding of the mechanistic aspects of telomere maintenance with and without telomerase.