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The architecture of Smc complexes and their functional roles in chromosome maintenance

Final Activity Report Summary - SMC COMPLEXES (The architecture of Smc complexes and their functional roles in chromosome maintenance)

Structural maintenance of chromosomes (SMC) complexes (including the complexes called cohesin and condensin), are proteineaous complexes that interact with DNA and have important roles in DNA repair, maintenance and function in almost all organisms. They are important for the condensation of chromosomes, and for the cohesion of the sister chromatids. Errors in their functions can have serious implications. For example, in humans, the correct function of the cohesin complex is important for accurate chromosome segregation during meiosis, and defects in cohesin can cause aneuploidy (trisomy or monosomy), the most common chromosome abnormality in humans causing diseases such as Down's Syndrome.

The SMC complexes are comprised of several protein parts. One or two types of SMC proteins and at least two accessory proteins (these include the kleisin family of proteins). It is currently thought that all SMC complexes function by a single conserved mechanical quality, whereby they assemble on DNA, forming a large proteinaeous ring than can encircle and hold together DNA. Evidence for this theory arises predominantly from genetic experiments in yeast; however the work funded here was focused at obtaining direct information about the SMC complexes molecular architecture using techniques such as electron microscopy and protein X-ray crystallography. This has been a long-term goal of the host laboratory (Dr Jan Löwes), in a collaborative effort with Dr Kim Nasymth's laboratory in Vienna, and more recently in Oxford.

The project proved more difficult than originally anticipated, many of the proteins were difficult to make in quantities suitable for carrying out experiments. Eventually excellent protocols were devised and we were able to prepare large amounts of protein and were able to successfully assemble complete SMC complexes in vitro ready for study. We discovered that SMC complexes are quite robust structures, and the various components form strong interactions with each other. We were able to obtain crystals for the first time of a part of the complex known as the 'hinge' from a heterdimeric form of the Smc complex (unique to higher eukaryotes). This region is where two Smc proteins interact together, and the structure of the heterodimer, comprised of SMC1 and SMC3 from mouse was determined by X-ray crystallography. This data revealed interesting points about the SMC proteins. The two proteins formed an interaction face with a 'hole' between them, which is intriguing. Furthermore, the hole has a conserved ionic surface charge. Genetic experiments investigating these qualities are currently being carried out in Kim Nasymth's laboratory, and should reveal interesting mechanical insights into SMC complex function.

We have also obtained crystals of ScpA (a bacterial kleisin-like accessory protein), for which no known structure has been resolved, and are working on obtaining data for determining the structure of ScpA, and ScpA in complex with the other accessory protein ScpB.