Synthetic chromosomes to decipher requirements for optimal transmission of DNA in yeast

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This project will use synthetic biology to uncover the features of DNA sequence, other than a centromere and DNA replication origin, required for "perfect" transmission of chromosomes in budding yeast mitosis. The length and organization of chromosomes influence their accurate segregation. Short (<50kb) linear chromosomes are poorly inherited through cell division. Increasing the length, or circularization, improves segregation fidelity: fragments of ~150kb show similar stability to endogenous chromosomes. The cohesin complex links newly duplicated chromosomes together and is essential for accurate segregation. We hypothesize that chromosomal DNA distant from centromeres is required to recruit and maintain sufficient cohesin to ensure chromosomes are robustly linked. Longer or circular chromosomes may recruit/retain more cohesin, increasing their stability. Transcription contributes to the loading and positioning of cohesin, suggesting that DNA sequence could promote segregation fidelity. We will design and assemble a library of artificial, non-essential minichromosomes made up of synthetic, transcriptionally silent DNA ranging from 26-150kb, in both circular and linear form. Using these unique tools, we will uncover the relationship between chromosomal size, circularization, segregation fidelity, cohesin recruitment and cohesion establishment. The requirements for building a specialized, cohesin-rich pericentromere, which directs and monitors chromosome segregation, will be determined. To test the idea that transcription and DNA replication influence cohesin position, cohesion and chromosome segregation, we will introduce synthetic transcriptional units and DNA replication origins. Finally, we will assemble an optimally segregating "designer" chromosome. This project will reveal fundamental requirements for chromosome segregation and provide tools for stable propagation of DNA in yeast.

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