Engineering synthetic mini-chromosomes to study chromosome segregation in budding yeast

Chromosomes need to be replicated and partitioned into daughter cells during cellular division. Failure to do so leads to aneuploidy, which has been linked to medical conditions such as cancer. Cohesin, a ring-shaped protein complex, holds the replicated sister chromatids together to ensure their accurate segregation. In Saccharomyces cerevisiae a large pool of cohesin is loaded at centromeres. Introducing the centromeric sequence to circular and linear DNA has been exploited to allow propagation of extra-chromosomal DNA. However, these extra-chromosomal DNA do not segregate as well as endogenous chromosomes and the reasons for this are unknown. Interestingly, small circular chromosomes are more stable than their linear counterparts. Furthermore, the stability of linear chromosomes increases with chromosome length. The DNA sequence itself appears to be important as linear chromosomes containing endogenous DNA are more stable than chromosomes derived from artificial DNA of similar lengths. During our project, we sought to identify cis-acting DNA elements, other than the centromere, that are important for chromosome segregation.

We have developed a synthetic biology approach to engineer a library of synthetic circular and linear chromosomes of various lengths containing unnatural DNA (designed to lack features of known elements such as transcription units). These chromosomes act as a “blank canvas” where candidate DNA elements are introduced to test their functionality. Our design, build and test cycle has allowed us to determine how chromosome length, circularization and DNA sequence influence chromosome segregation and the minimal requirements for building the optimal synthetic chromosome. Our latest findings will be presented.