tpohl

Misregulation of DNA replication leads to increased genomic instability. DNA replication initiates at specific genomic sites known as origins of replication (origins). Origins vary in both efficiency and time of activation (early S-phase vs late S-phase). Mechanisms governing origin regulation remain largely unknown. Centromeres in Saccharomyces cerevisiae replicate early yet the biological significance of this is unknown. We hypothesize that centromeres either promote early activation of nearby origins or selectively reside in early replicating genomic regions. We are exploring these possibilities by creating strains in which the centromeres are relocated to regions harboring late activated origins. Genome wide origin replication times in the modified strain can be compared to wild type using modified Meselson-Stahl density transfer assay. Shifting of an origins replication time from late to early relative to wild type suggests that the centromere promotes early activation of nearby origins. Mutants deficient in kinetochore formation or centromere chromatin structure can be tested to see if centromere-mediated early activation of late origins is abolished. Alternatively, failure to shift replication timing suggests that the centromere does not actively affect replication timing of nearby origins. However, the modified strain can be tested in a growth competition assay to see if early centromere replication confers a growth advantage.