A genetic analysis of telomerase translocation and telomere function in L. lactis
Underwood, Dana Elizabeth Hager
MetadataShow full item record
Telomeres are DNA-protein complexes that protect the ends of linear chromosomes. The non-genic DNA is typically composted of repetitive T/G-rich sequences, and maintained by telomerase, a specialized reverse transcriptase. In the yeast K. lactis, the telomeric repeats are 25 base-pairs in length and copied from a 30 nucleotide template within the telomerase ribonucleoprotein complex. In order to examine the functional regions contained within the telomere and the telomerase template, strains were constructed such that each contained a single base change in the region of the TER1 gene encoding the telomerase template. Mutations were made at all 30 template positions, and the effects were initially examined by measuring the lengths of the telomeres in the mutant yeast. Telomeres were also cloned to examine the types of mutant telomeric repeats added by telomerase. Typically, the mutations were incorporated as single base-changes in the newly synthesized telomeric repeat. Mutations leading to telomere elongation, telomere shortening, delayed telomere elongation, and telomeric repeats of aberrant sizes were identified. Mutations causing each phenotype were often confined to discrete regions of the template. Mutations leading to telomere elongation and aberrantly sized telomeric repeats were examined further. Extreme telomere elongation was found to be independent of RAD52-dependent recombination. However, the formation of the large amount of extrachromosomal and single-stranded telomeric DNA observed in these mutants was found to be dependent on RAD52. Subtelomeric recombination was also found to be elevated in these mutants. Mutations in the ends of the telomerase template led to the synthesis of telomeric repeats that were not 25 base-pairs in length. By examining the telomeric repeats in these mutants, it was determined that mutations which disrupted the base-pairing between the telomere and the template that occurs prior to the synthesis of new telomeric repeats caused the telomere to align with a different region of the template. Mismatches, therefore, were typically neither extended nor removed by a nucleolytic activity. It was also determined that a region outside of the template might play a role in stabilizing the base-pairing interactions between the telomere and the template prior to the synthesis of new telomeric repeats.