Structural Biochemistry of Meiotic Double Strand Break Control

• Applicant: Dr. John Weir
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 445087119

Sexual reproduction requires the generation of germ cells (eggs and sperm). Germ cells have half the number of chromosomes from the parent, so that two germ cells can fuse to produce a cell with a complete complement of chromosomes. The accurate segregation of parental chromosomes into germ cells is called meiosis.In order for the chromosomes to be accurately sorted during meiosis, they need to be linked to one another. One linkage comes during the preceding DNA replication phase, but the other, meiosis specific linkage, needs to be introduced.Meiosis specific chromosome linkages match up similar, so called homologous, chromosomes. In order for similar chromosomes to find one another, the DNA first has to be broken. DNA breaks are made by a machinery made up of many different proteins. Since DNA breaks can be very dangerous for a cell, they are tightly regulated.Control of meiotic DNA breaks occurs on three levels, namely temporally, spatially and numerically. This control is achieved by modifying the DNA break protein machine, or proteins that wrap up DNA. These modifications occur in the form of ‘post-translational modifications’, which include the attachment of chemical groups that are added to proteins which can alter their activities.This proposal aims to look in detail at how meiotic DNA break formation is controlled. We will take a novel approach of removing all proteins of interest from the cell, and studying them ‘in vitro’; in a test tube. Initially we will use proteins from the budding yeast model system, where meiosis is best understood. Importantly, budding yeast carries out meiosis in a way that is very similar to other organisms, including humans. We propose to use our understanding of yeast meiosis to look more closely at mammalian proteins. Our in vitro approach allows us to control the environment that the proteins are in, and to add and remove other components of the system. Importantly, we will be able to either add, or remove specific post-translational modifications, and determine that effect they have the DNA break machinery.We will combine our in vitro studies with structural biological methods, to visualize our proteins of interest in incredible detail. By using a combination of X-ray crystallography and Cryo-electron microscopy, we will study the proteins and protein complexes in such high-resolution that we will learn the function of individual amino acids, and individual post-translational modifications. Such detail will allow us to design more sophisticated future experiments based on our high-resolution structures.In summary, the unprecedented level of detail that we will obtain from this study will give new insights into the control of meiotic DNA break formation. This will have important implications for both basic research, but also in understanding what can go wrong during meiosis. In humans, a failure to carry out a correct meiosis, can lead to adult infertility or chromosomal diseases in children.

• DFG Programme: Research Grants