Localisation, regulation and functional significance of the SMC-loading complex during eukaryotic cell division.

The strucural maintenance of chromosome (SMC)-complexes organise and stabilise higher order chromosomal conformations during normal cell division, e.g. condensation of chromosomes cell division or generation of inactive chromatin, cohesion of newly replic ated sister chromatids and stabilisation of replication factories during DNA replication. Furthermore, these complexes are also involved in the generation of nuclear architecture, gene regulation and DNA repair. With all these diverse and pivotal functi ons in mind, it is intriguing that the chromosomal association of these complexes are controlled by a common loading factor, which in yeast consists of the Scc2 and Scc4 proteins. In mammals, these proteins are referred to as NIPBL and SCC4, respectively, and mutations in the NIPBL-gene causes Cornelia de Lange (CdLS)-syndrome, affecting approximately 1 in 10000 live births. In this project we will investigate the post-translational regulation of yeast Scc2 and Scc4 by mass-spectrometry, using purifying epitope-tagged proteins. We will then characterise the functional significance of these mutations, and its functions in chromosome dynamics during the cell cycle and in response to DNA damage. We will also investigate the localisation and function of th e mammalian SMC-loading complex during meiosis, by visualising these proteins on murine meiotic chromosome spreads. Since the SMC-complexes perform meiosis-specific functions and display different binding patterns during meiosis, the functional significan ce of the SMC-loading complex is of great interest. Finally, we will also investigate the nuclear localisation of the SMC-loading complex in cells from CdLS-patients and familial controls during the cell cycle and in response to DNA damage. We will also follow up recent reports from others, indicating that CdLS may be caused by dysfunctions in gene regulation brought about by alterations in nuclear architecture.