Use of synthetic DNA to study centromere function in Saccharomyces cerevisiae

The function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail. Twelve of the sixteen S. cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified. This sequence consists of a central region, conserved centromere DNA element II (CDE II), which is 78-86 bp in length, greater than 87% A+T-rich, and tends to be arranged in runs of As and runs of Ts. The central region is flanked on one side by a highly conserved 8 bp sequence (CDE I) and on the other side by a highly conserved 25 bp sequence (CDE III) which contains partial inverted dyad symmetry around a central C/G base pair. Mutational analyses have been used to determine the importance of the consensus sequences to centromere function. A protein which binds to the CDE I sequence and at least one protein that binds to the CDE III sequence have been identified. The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation. In this study, totally synthetic DNA was used to create centromere mutants that could not have been made easily by any other method. Functional analysis of these mutants have confirmed and extended the findings of other workers. First, the results provide supporting evidence for the idea that the centromere consensus sequence is sufficient to confer wild-type mitotic and meiotic function to a replicated plasmid or chromosome. Second, they support the idea that the highly conserved base pairs in CDE III are important for maintaining the symmetry and functionality of CDE III. Third, the results suggest that a protein important for proper mitotic centromere function binds to CDE III and interacts directionally with something located at or near CDE II. Fourth, they confirm the importance of the A+T-richness of CDE II for proper mitotic and meiotic centromere function, and they provide evidence that the ability of CDE II to form a bend may also be important for both mitotic and meiotic centromere function. Finally, one mutant functioned better on plasmids than on chromosomes, a finding that has interesting implications for chromosome structure and function