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

Cell division is the process by which organisms reproduce themselves. During this process, the genomic DNA, carried on chromosomes, must be replicated and equally distributed to the daughter cells. Errors in this process produce cells with the wrong number of chromosomes. This is associated with cancer and causes birth defects and infertility. Understanding how this process works is therefore medically important. Many of the features of chromosome segregation are common between humans and very simple single-celled organisms, such as baker's yeast. Yeast has a long history as a discovery tool in mechanisms of chromosome segregation since it is easy to grow and manipulate genetically. Furthermore, yeast has advantages over other microorganisms such as bacteria for some industrial applications. Yeast is non-toxic, easy to manipulate and, since the cellular machinery is closer to that of humans than bacteria, yeast cells are more likely to recapitulate the production of human biomolecules. To achieve this, yeast must faithfully transmit the DNA carrying the instructions to produce these biomolecules over many generations. However, we do not yet have a complete understanding of how DNA is transmitted accurately to daughter cells during cell division. This knowledge will not only help us understand what goes wrong in human diseases, such as cancer, but will also help us design better DNA carriers for biosynthesis.

This project will take a "synthetic biology" approach to address this problem. Natural yeast chromosomes are densely packed with important features for cells to grow and flourish. These include genes which are transcribed and translated to make protein products that build the machinery for chromosome segregation. Therefore, it is difficult to dissect out the properties of the DNA sequence itself that promote chromosome segregation, the aim of this study. To avoid these problems, we will chemically synthesise DNA and build chromosomes from scratch, first in a computer, then in a test tube and finally in yeast. These chromosomes will not be required for cell growth and will not carry any genes, allowing us to examine the role of DNA sequence and activity. We know already that chromosome length and circularization influences its segregation. We will build chromosomes of different lengths in both linear and circular form. We will then examine the ability of these synthetic chromosomes to recruit a key factor, called cohesin, that sticks chromosome pairs together. We expect chromosomes with poorer segregation to recruit less cohesin. Next, we will test the idea that the act of gene expression itself helps to recruit cohesin and improve chromosome segregation. We will add genes encoding protein products without a cellular function onto the minichromosomes and test the effect on cohesin recruitment and chromosome segregation. Endogenous chromosomes assemble a cohesin-rich domain, called the pericentromere, around the point at which pairs of chromosomes are attached to the spindle that will pull them apart. We will use the synthetic minichromosomes to identify the features important for pericentromere formation. Our final objective is to use the knowledge gained in this study to build a "designer" chromosome with "perfect" chromosome segregation.
Overall, this study will provide fundamental biological knowledge of the sequence features of DNA which ensure its accurate transmission during cell division. This research will be useful in the future in the design of DNA carriers for industrial applications. Furthermore, an important priority for this project is engagement with the public to encourage discourse around the new scientific area of synthetic biology and its potential.

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.

This research will have four broad impactful outcomes. First, it will lead to important insights into fundamental mechanisms of chromosome segregation that have been elusive so far. Second, it will promote and propagate the use of synthetic biology as a research tool. Third, it will produce a designer chromosome that can be adapted for academic and industrial applications. Fourth, by embedding a public engagement project "Big ideas, tiny tools" within the research programme, it will provide much-needed discourse around the discipline of synthetic biology.

Together these outcomes will benefit varied groups of people. First, the public will benefit through our engagement project "Big ideas, tiny tools" which will target adult audiences that may typically not engage with science. These include library book clubs and prisoners. These groups of people will benefit from being exposed to new ideas and we will benefit from hearing their opinions. We are particularly keen to hear from individuals sceptical about genetic engineering and understand the reasons for this concern. Second, the wider academic community will benefit both from the generation of new tools, protocols and reagents that they can employ in their own research, and from the fundamental knowledge gained which will promote further research in this area. Third, undergraduates will benefit from the introduction of the concepts and findings surrounding this research into their lectures, informing them of the very latest areas of research and enhancing their employability. Fourth, the UK economy will benefit as this project will help to consolidate the UK's position as a leader in synthetic biology and attract talent to pursue this exciting new area of biology. Fifth, industry will benefit by the availability of a designer chromosome with perfect segregation. Assembly of biosynthetic pathways on a stably maintained DNA carrier would be expected to increase productivity as it would allow culturing over many generations without loss of yield. During the later stages of the project we will engage with industrial partners and apply for a CASE studentship and pump priming funding to explore the possibility of using our tools/protocols for biosynthesis in yeast and beyond.

Our vision to achieve these impactful outcomes ranges beyond publication in scientific journals and attendance at scientific conferences. We will engage with organisations that connect industry and academics to raise awareness of our work and its potential applications. We will work with public engagement teams in the Wellcome Centre Cell Biology and (BBSRC-funded) UK Centre for Mammalian Synthetic Biology to deliver an innovative programme to the public. As the project progresses, we will incorporate developments into these activities and remain committed to ensuring that maximum benefit is derived from our work by as many people and organisations as possible.