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EASTBIO Understanding cohesion establishment at DNA replication origins using a synthetic biology approach

  • Full or part time
    Prof A Marston
  • Application Deadline
    Sunday, January 05, 2020
  • Competition Funded PhD Project (Students Worldwide)
    Competition Funded PhD Project (Students Worldwide)

Project Description

Second Supervisor: Prof Anne Donaldson, University of Aberdeen

During cell division, chromosomes must be replicated and then evenly distributed to daughter cells. Defects in this process will generate daughter cells with the wrong number of chromosomes, which causes birth defects and is a characteristic of cancer cells. Accurate chromosome segregation begins with DNA replication which creates two identical sister chromatids that are tightly cohered together by the cohesin complex. During mitosis, cohered sister chromatids attach to microtubules from opposite poles and cohesin resists their separation. Once all chromosomes have properly attached to microtubules, cohesin is destroyed, triggering the even segregation of sister chromatids to opposite poles. The establishment of sister chromatid cohesion is therefore critical for accurate chromosome segregation. This project aims to understand how cohesion establishment is coupled to DNA replication by taking a “bottom up” synthetic biology approach in budding yeast. In this organism, DNA replication initiates from a number of sequence-defined origins, spread throughout the genome. We have used synthetic biology methodology to design and build a library of artificial mini-chromosomes with a single DNA replication origin close to the centromere, which is a strong cohesin-loading site. These chromosomes also lack transcriptional units and therefore serve as a “blank canvas” to test the influence of particular DNA sequences on replication, cohesion and segregation, without the confounding effects of other chromosomal processes, such as transcription.

Aim 1: Determine how the number, timing and position of DNA replication origins influences cohesion establishment
To determine the sites of DNA replication initiation and dynamics on the artificial minichromosomes, replication profiles will be determined using next generation sequencing methods. Next CRISPR-Cas9 technology will be used to re-locate origins, add additional origins, or alter the timing of origin initiation and replication profiles generated. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) will determine the positions of cohesin. Live cell imaging and chromosome loss assays will be used to understand the effects of changes in the replication profile on chromosome segregation. Together, this will reveal the relationship between replication origin position and cohesion establishment, while providing training in next generation sequencing methods and analysis and advanced microscopy methods.

Aim 2: Understand the nature of cohesin associated with synthetic chromosomes
Cohesin is loaded prior to DNA replication and becomes functional in holding sister chromatids together only after DNA replication. Artificial minichromosomes that can load cohesin, but fail to convert it into cohesion, provide an opportunity to decipher the modifications on cohesin that make it cohesive. To address this, the synthetic minichromosomes with cohesive vs. no cohesive cohesin will be purified from cells biochemically and associated proteins, together will their post-translational modifications, will be identified by mass spectrometry. This will provide training in proteomics and mass spectrometry analysis and reveal how sister chromatids are held together in preparation for their accurate segregation during mitosis.






Funding Notes

The “Visit Website” button will take you to our Online Application checklist. Complete each step and download the checklist which will provide a list of funding options and guide you through the application process. Follow the instructions on the EASTBIO website (you will be directed here from our application checklist), ensuring you upload an EASTBIO application form and transcripts to your application, and ticking the box to request references. Your referees should upload their references using the EASTBIO reference form, in time for the 5th January deadline so please give them plenty of time to do this by applying early.

References

(1) Müller CA, Hawkins M, Retkute R, Malla S, Wilson R, Blythe MJ, Nakato R, Komata M, Shirahige K, de Moura AP, Nieduszynski CA. (2014) The dynamics of genome replication using deep sequencing. Nucleic Acids Res. doi: 10.1093/nar/gkt878
(2) Hinshaw S*, Makrantoni V*, Harrison S† and Marston AL (2017) The kinetochore receptor for the cohesin loader. Cell 171, 72-84.
(3) Paldi F, Alver B, Robertson D, Schalbetter SA, Kerr A, Kelly D, Neale MJ, Baxter J and Marston AL. Convergent genes shape budding yeast pericentromeres. doi: https://doi.org/10.1101/592782

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