Re-replication of CEN9 Causes Aneuploidy

Eukaryotic DNA replication utilizes many mechanisms to prevent the re-replication of any genomic segment. Such re-replication at high frequencies is known to lead to cell death presumably due to genomic instability. The consequences of DNA re-replication leading to this instability has not been analyzed. We  hypothesize that inappropriate re-replication of a chromosome centromere will disrupt mitotic segregation of the chromosome and result in chromosome aneuploidy. To test this in the budding yeast Saccharomyces cerevisiae, we conditionally induced localized re-replication of CEN9, the centromere of Chromosome 9 (Chr 9).  We followed the distribution of the Chr 9 during cell division using a copy number reporter that affects the color of yeast cells depending on the number of Chr 9 in the cell; cells with no copies are white, one copy pink, and two or more copies red.  After inducing transient re-replication of CEN9 we plated individual cells and allowed them to grow into whole colonies.  Most cells properly distributed 1 copy of Chr 9 to each daughter cell (1:1 segregation) to generate pink colonies.  Cells that aberrantly distributed two copies of Chr 9 to one daughter cell and none to the other (2:0 missegregation), lead to red/white sectored colonies.  Preliminary results indicate an average increase of 1.53% in red/white sectored colonies indicative of a chromosomal missegregation event. We will confirm that the cells from these colonies have an incorrect copy number of Chr 9 using array comparative genomic hybridization, which directly monitors the copy number of every segment of the genome.