Discovering the molecular function of Srs2 helicase during meiosis

Fertility of sexually reproducing organisms depends on their ability to create gametes by a form of cell division called meiosis. Organisms that do this have two sets of each chromosome, one set inherited from the mother, and one set inherited from the Father. During meiosis parental chromosomes are shared out so that the gametes inherit only one copy of each chromosome. This part of meiosis is called chromosome segregation. To segregate chromosomes successfully they must be organised in a special way in the nucleus of the meiotic cell. Also, parts of the maternal and paternal chromosomes swap places so that the chromosomes in the gamete are effectively a patchwork of grandparental genetic material. The process of swapping around genetic material is called recombination. Without the chromosome reorganisation and recombination, chromosome segregation would fail in meiosis, causing either sterility or leading to genetically abnormal offspring.

This project studies the role of an important protein that we believe helps cells to reorganise chromosomes and swap around genetic material. The protein is called Srs2. Srs2 is needed in daily life to help cells avoid DNA damage that occurs continuously in all cells due to both internal and external factors (such DNA replication and chemical attack). Our preliminary experiments indicate that Srs2 also influences the ability of cells to do recombination in meiosis. When the protein is mutated the cells cannot do meiosis very well and there is a significant reduction in fertility.

In this proposal we plan to study the DNA molecules involved in recombination during meiosis. We will also study carefully the location of the Srs2 protein to understand where it is in the nucleus and which other proteins it interacts with. By examining other proteins in cells with mutated Srs2 we will be able to determine how Srs2 exerts it influence. We have hypothesised that this is by changing the ability of other important recombination proteins to bind DNA. If we are right then this will significantly increase our understanding of how meiosis works and fertility is maintained.

We will conduct these studies in an experimental yeast organism. At the cellular level yeast is surprisingly similar to more complex sexually reproducing organisms like humans. In fact yeast is an excellent model system for what happen in human meiosis, and most genes known to be important for human fertility were discovered in yeast experiments. Humans have a protein that is similar to Srs2 (called hFbh1) so the information we gather about Srs2 function will tell us about what happens in humans too. It is only by doing these experiments in model organisms such as yeast that we can plan and later perform the right experiments model animals, such as mice, to get an even better idea of what happens in humans. The studies will tell us, for example, whether or not it is worth investigating hFbh1 to understand and explain cases of human infertility.

Sexually reproducing organisms create haploid gametes by segregating parental homologous chromosomes through meiosis. In most organisms successful segregation of homologues requires crossing over, which follows chromosome pairing, synapsis and homologous recombination. Failure or perturbation of any of these can impinge on chromosome segregation and reduce fertility.

This project aims to test ideas about the meiotic role of Srs2, a DNA helicase in Saccharomyces cerevisiae. Srs2 and functionally related proteins (FBH1 and RecQ helicases) have conserved helicase function at the heart of genome stability in mitotic cells. Srs2 also has DNA translocase activity, which can strip Rad51 from single-stranded DNA, so impeding strand invasion reactions during homologous recombination.

We have found that cells expressing the srs2-101 allele (which cannot hydrolyse ATP) have reduced spore viability. We discovered that the mutation causes faster turnover Spo11-induced DNA double-strand breaks (DSBs), the crossover frequency is reduced and there is a delay is dissolution of the synaptonemal complex (SC).

We plan to test the idea that normally in meiosis Srs2 acts upstream of Sgs1 to reduce Rad51/DNA filament formation to favour binding of the meiosis specific RecA orthologue, Dmc1. We suggest this is important to ensure proper crossover formation, perhaps in part by increasing the chance of interhomologue strand invasion events over intersister strand invasion.

We will test our ideas using both the srs2-101 allele and the allele srs2 1-860, which cannot interact with Rad51. We will further characterise recombination intermediates in mutant strains, including an assay of joint molecule formation. ChIP will be performed to determine if the binding of Rad51 and Dmc1 near DSB sites is directly affected by Srs2 function. In cytological experiments we will look at the location of Srs2 in the nucleus, and determine with which relevant proteins it colcalises.

This proposal centres on developing a better understanding of how cells do meiosis, which is of fundamental importance to all sexually reproducing organisms. Learning about meiosis provides benefits by impacting on the following BBSRC research priorities:

Lifelong health and wellbeing, because issues of increasing infertility with age are of major concern in developed countries. A deep understanding of meiosis is also important to increasing our understanding of how cells repair DNA damage, which contributes both to cancer and ageing. The particular protein we will study is very important for avoiding cancer and similar proteins are involved in the ageing process, and when faulty both cancer and premature ageing can result.

Food Security through Animal health and Livestock, which are dependent on maintenance and management of fertility. Furthermore, there is potential for development of non-invasive contraception for animals by targeting meiotic proteins with specific inhibitors.

Food Security through crop science. The main barrier crossing desirable genes into crops is the mechanism built into meiosis that prevents recombination between chromosomes of different species. This project directly addresses issues of how crossing over is regulated at a molecular level, and therefore the output of this work is potentially of importance to crop breeders looking for ways to improve hybrid fertility.

The employment of a postdoctoral scientist and a technician will contribute to the national and local economy. The scientific training they receive will enhance future employability prospects and provide them with the skills needed to be decision makers and creative thinkers. The speaking, reporting and thinking skills staff will develop are transferable to other employment sectors and the self-confidence they provide will render the staff highly competitive in the broader job market.

The work also has significant impact beyond those directly involved in the science. It will provide employment for indirect contractors and suppliers who maintain equipment and supply and deliver reagents.

Beneficiaries also include the general public. Benefits to school children will be derived from the PI with the help of staff employed on the grant, running workshops on science directly related to this project during science week. In the past the PI has run 1 hour workshops with junior school children in which they learnt about chromosomes and DNA in reproduction. These will be repeated with a new cohort of children. The PI will offer to speak at the local Café Scientifique, which is held monthly in Sheffield, and generally attracts a lay audience of around 80 adults. They will benefit educationally from hearing about research, what sort of questions we ask and how we answer them. With specific examples of research data provided such talks not only inform but can inspire adults to become more involved in science, sometimes changing their career path altogether.

Close to The University of Sheffield is Western Park a natural history and science museum. The PIs laboratory has previously contributed to a permanent video display there and will offer further contribution, including providing workshops events to children.

The above contribute to the nation's general health and wealth by increasing employability and science awareness, which in turn feeds creativity. They will all be delivered within the lifespan of the project.

There is also a long-term future benefit to society based on further discoveries and potential improvements in medical treatments. The work directly addresses issues important to genome stability and fertility and the basic science knowledge derived will in the future will be informative to more applied translational science.