UC San Diego

As cells prepare to divide, their genetic information needs to be duplicated. In order to do so, many proteins work together into the replisome that faces many hurdles and difficult-to-replicate areas. Among which, the antisense nature of the lagging strand itself results in the end-replication problem, a gradual erosion of the extremities of eukaryotic linear chromosomes each cell cycle. Extreme shortening of telomeres, the ends of chromosomes, can trigger DNA recombination, chromosome fusion and overall increase the chance of deleterious damage that potentially threatens viability.DNA polymerase alpha-primase is the main complex responsible for antisense lagging strand replication. It has been argued to closely interact with t-RPA (telomeric Replication Protein A), a telomere-specific DNA binding complex. This heterotrimer, made out of Cdc13, Stn1 and Ten1 and also known as the CST complex, is known to be an essential player to recruit the enzyme telomerase. Telomerase is able to generate de novo telomeric sequences to maintain the ends of chromosomes over cell cycles. This dissertation is the result of my focus on Pol12, non-catalytic subunit of polymerase alpha. This protein, although essential, has had very little insights about its function. By applying a genetic screening assay adapted in our laboratory, we uncovered several clusters of residues on the surface of the protein in Saccharomyces cerevisiae. These clusters have distinct DNA replication and telomere homeostasis-related phenotypes. Most notably, a subset of these mutants is synthetically lethal when combined with a t-RPA defect. Those same mutations show a decreased level of interaction between Pol12 and its hypothesized interacting t-RPA subunit, Stn1. It is expected that this loss of interaction impairs the post-replicative C-strand fill-in process of telomeres. This study further confirmed how the two complexes seem to work closely, both genetically and physically.