EASTBIO: What causes the long duration of pre-meiotic S-phase?

Supervisors:

Dr Alexander Lorenz (University of Aberdeen) https://homepages.abdn.ac.uk/a.lorenz/pages/index.html

Professor Adele Marston (University of Edinburgh) http://marston.bio.ed.ac.uk/

Professor Anne Donaldson (University of Aberdeen) https://www.abdn.ac.uk/ims/profiles/a.d.donaldson

The cell cycle, a recurring cellular process consisting of DNA replication followed by cell division, produces two identical daughter cells from a single precursor cell. Its accuracy is of the utmost importance to maintain genome integrity, and thus cell health (cell cycle dysregulation is a hallmark of cancer). The cell cycle phase during which DNA is replicated is called S-phase.

The duration of the cell cycle (and S-phase) is similar for cells of the same species, given similar nutritional conditions and provided that DNA integrity is not compromised. Intriguingly, the cell cycle progression leading to meiosis (the cell division producing gametes) is however much slower than a mitotic cell cycle. Two cell cycle stages are especially elongated: the pre-meiotic S-phase and meiotic prophase I last three-times longer than their mitotic counterparts. Meiotic prophase I requires more time to complete due to the requirement for chromosome pairing, recombination, and synaptonemal complex formation. However, the substantial extension of pre-meiotic S-phase remains unexplained, and its underlying causes are still enigmatic.

This project will explore and elucidate the mechanism and regulation of pre-meiotic S-phase duration, addressing the following research objectives:

1) Determine origin usage and fork speed in pre-meiotic S-phase

Pre-meiotic S-phase extension could be explained either by fewer replication origins being fired or by replication forks moving more slowly than during mitotic S-phase. Genome-wide replication profiling (data-driven biology) of the baker’s yeast Saccharomyces cerevisiae by next-generation sequencing of mitotic vs pre-meiotic S-phase cells will determine differences in origin usage between these cell cycle stages.

Fork speed will be characterized by DNA combing combined with fluorescence in situ hybridization (FISH) to assess whether the same early- and late-initiating replication origins are used in pre-meiotic S-phase as in mitosis, and to determine whether fork speed is similar between mitotic and pre-meiotic S phase.

2) Are characteristics of pre-meiotic S-phase evolutionarily conserved?

The fission yeast Schizosaccharomyces pombe is only distantly related to baker’s yeast, and thus represents a great model organism for comparative analysis. DNA combing/FISH will reveal whether differential fork speed/origin usage during meiosis is conserved.

3) What modulates behaviour of origins in pre-meiotic S-phase?

Pre-meiotic S-phase fork speed/origin usage clearly require specific regulation operating differently from the normal mitotic cell cycle.

This objective will employ mutant analysis to identify whether CDKs (cyclin-dependent kinases), DDK (Dbf4-dependent kinase), and/or the cohesin complex are the regulatory components that specify pre-meiotic S-phase length.

This project will employ a variety of state-of-the-art techniques to elucidate how and why pre-meiotic S-phase is so much longer than mitotic S-phase.

The successful candidate will be trained in a variety of genetics, genomics, and molecular cell biology techniques to exploit new ways of working to elucidate a fundamental cellular process crucial to cell health and fertility. This project will allow the student to gain skills in data-driven biological research by generating a comparative database of origin usage of mitotic vs pre-meiotic yeast cells via next-generation sequencing; additional training in DNA combing, fluorescence in situ hybridization, and comparative yeast genetics will be provided.