Structure and mechanism of the Swr1 histone exchange complex

DNA is a fragile molecule and if damage to DNA is not repaired, it can lead to cancer. To help protect the DNA, the genetic material in a human cell is wrapped around a protein core (comprising two copies each of four histone proteins) into nucleosomes. These nucleosomes are then packaged to form chromatin. Chromatin is more stable than naked DNA, protecting it from damage. It also serves to compact the DNA so that it can fit into the nucleus of the cell. However, biological processes such as DNA replication and transcription require unpacking of the DNA to expose the DNA molecule itself making it more susceptible to DNA damage. Repair of this damage also requires the DNA molecule to be laid bare to allow access of repair enzymes. Cells contain a variety of enzyme complexes whose functions are to process chromatin in a variety of ways including shifting the nucleosomes around (remodelling) and chemical modifications that are part of cellular signalling systems. Another group of chromatin modifying complexes exchange the proteins in the histone core for different variant histones that are used as signals to promote further processing. SWR1 complex (and its human equivalent SRCAP complex) is an example of one of these histone exchange complexes. It comprises 14 different protein subunits and it replaces the H2A histone with a variant called Htz1 (H2AZ in humans). This is a part of the DNA damage signalling pathway that eventually leads to recruitment of DNA repair enzymes. This proposal seeks to understand the molecular structure of the yeast SWR1 and human SRCAP complexes and to determine the mechanism by which these complexes use ATP to promote histone exchange. Using a combination of X-ray crystallography and recent advances in cryo-electron microscopy we will determine high resolution images of the complexes to understand their molecular architecture at atomic detail. These structural studies will suggest ideas about mechanism that we will test using biochemical tools that we have developed over the last few years.

Chromatin is a compacted form of packing of DNA in cells that, although it promotes stability of the genetic material, causes problems with gaining access for biological processes such as replication and transcription. Access to the DNA is also required for repair. Cells contain a variety of protein complexes that remodel chromatin, to slide nucleosomes away from regions where access is required or to replace them after these processes have been completed. However, other complexes have different roles in signalling to recruit proteins for different functions. These include complexes that covalently modify histones (e.g.by acetylating lysines in histone tails) and others that physically exchange histones for other histone variants. The 14 protein (1MDa) yeast SWR1 complex (and its human equivalent SRCAP) catalyses the ATP-dependent exchange of histone H2A/H2B dimers for Htz1/H2B (or H2AZ/H2B) dimers. This acts as a signal for downstream processing and recruitment of DNA repair factors but also plays a poorly understood role in regulation of transcription. We have prepared milligram quantities of recombinant SWR1 (and SRCAP) complexes in insect cells and will use this material for high resolution structural studies by X-ray crystallography and cryo-electron microscopy. This structural information will suggest mechanisms for histone exchange that we will explore further using biochemical analysis exploiting a number of different tools we have prepared in the last few years. By combining these studies we hope to gain greater understanding of this important process in DNA repair.

This research will provide information on molecular mechanisms which will underpin the design and interpretation of studies on biological function and cancer development. The proposed programme is basic research of potential value to the commercial private sector, specifically the biotechnology and pharmaceutical industries, to inform and guide the design of novel anti-cancer compounds. Cancer is a major medical concern and this proposal aims to lead to a greater understanding of chromatin regulation and will allow us to evaluate the potential of inhibitors of DNA break repair in humans that would may open up new opportunities for development of cancer drugs.

Another area of impact for this research will be in the training and career development of young research scientists. I have been very fortunate in the high level of talent and enthusiasm shown by the young scientists who have worked with me and I believe my laboratory has provided them with an excellent training environment. Past pre- and post-doctoral members of my laboratory include three UK university professors, two pharmaceutical industry researchers, one research charity grant board coordinator, several group leaders in labs abroad, plus several post-doctoral research workers in posts both in the UK and abroad who are on career trajectories to run their own research groups in due course.