DNA Recruitment, Regulation & Function of SMC5/6

点击翻译按钮获取中文摘要

Complex regulatory mechanisms control chromatin structure. Hereby protein complexes of the SMC family (cohesin, condensin and SMC5/6) play essential roles. Cohesin and condensin form large rings which embrace DNA duplexes. While cohesin keeps sister chromatids together - from the moment they emerge through DNA replication till the onset of cell division - condensin facilitates chromosome compaction and accurate segregation of one copy of the genetic material to the daughter cells. In contrast, the exact function of SMC5/6 remains unknown. Inactivation of SMC5/6 leads to an immediate cell cycle arrest in mitosis or inaccurate transmission of the genetic material to the daughter cells, respectively. Consistently, compromised SMC5/6 function accelerates tumorigenesis in animal models.Here we propose to study SMC5/6 function in Xenopus egg extracts. Our preliminary results show that SMC5/6 is recruited to plasmid substrates in a replication-dependent fashion. Importantly, we detect plasmid-bound proteins with newly-developed, unbiased proteomic methods. This allows us to accurately quantify all SMC5/6 subunits as well as cohesin and all DNA replication factors.Using this unique system we will address the following questions:1) When and how is SMC5/6 loaded onto DNA?We will monitor protein recruitment to DNA during normal and perturbed DNA replication. We will determine the cellular network regulating the physiological recruitment of SMC5/6 to DNA by identifying the relevant DNA substrates and loading complexes. We will test whether the SMC5/6 proteins form ring-like structures that mediate topological binding of the DNA. We further propose experiments to gain insight into how the distinctive structures of SMC5/6 and its ATPase, sumo and ubiquitin E3 ligase activity relate to its function. 2) What are the functions of SMC5/6?We will generate reagents for the depletion of SMC5/6 or its loading factors from Xenopus egg extracts as well as recombinant proteins for rescue experiments. Using depleted extracts with defined plasmid substrates we will analyze DNA replication and repair intermediates using a variety of established assays. These experiments will directly reveal a possible function of SMC5/6 in the decatenation of replication products or in distinct steps of defined DNA repair pathways.By combining a biochemically tractable system with defined DNA substrates and unbiased, ultra-sensitive proteomic methods, we will for the first time be able to correlate the temporal appearance of DNA intermediates with the assembly of molecular machines involved in the DNA transactions. We anticipate that these methods will provide unprecedented insight into the function and action of the hitherto enigmatic SMC5/6 complex.