Structure of the Smc5-6 DNA repair and chromosome maintenance protein complex
Lead Research Organisation:
University of Sussex
Department Name: Sch of Life Sciences
Abstract
The accurate inheritance of the genetic material (DNA) is key to the survival of all organisms. DNA has to be copied (replicated) before cells divide but obstacles such as damaged DNA bases can lead to problems during the copying process. Cells have developed different ways to remove such obstacles (repair), and if the DNA damage cannot be removed it can be bypassed, either during or immediately after DNA copying (tolerance). Failure of repair or tolerance can result in errors when the DNA is copied. These errors cause permanent genetic changes, which in turn can result in increased cancer incidence. Many proteins are involved in repair and tolerance. One of these, Smc5-6, which we have been studying for several years, is a big ?protein complex? with eight components. We propose to combine the expertise of our four research groups to determine the 3-dimensional structure of this complex. This will enable us to understand how the different components fit together, what they each do, and how they help to fulfil the functions of the protein complex in the cell. This will help us to understand how Smc6 acts to protect us from the problems caused by DNA damage during DNA copying. This work has implications for the development of cancer and aging in the general population.
Technical Summary
The SMC protein complexes, including cohesins and condensins, are required for maintenance of higher order chromosome structure and segregation. The Smc5/6 complex, the least well understood of the three SMC complexes, is required for DNA repair, rDNA stability and telomere maintenance. It consists of 8 subunits, which form three sub-complexes: the core Smc5 and Smc6 proteins with the SUMO ligase Nse2; Nse1-Nse3-Nse4, which includes the RING finger putative ubiquitin ligase Nse1; and Nse5-6. A further protein, Rad60 interacts transiently with the complex.
We wish to understand the in vivo functions of the Smc5/6 super-complex in terms of its biochemistry and its structure. In particular we wish to define the functional contributions of the individual subunits, determine how they assemble in the functional complex and sub-complexes, and elucidate the structural basis for regulation and coordination of the ATPase, SUMO ligase and ubiquitin E3 ligase activities, which the complex incorporates.
Towards this end we will develop expression and purification strategies for the assembled complex ex vivo from S.pombe, and the human and S.pombe complex, sub-complexes and individual proteins by recombinant expression in insect cells. We will use single-particle electron microscopy to define the overall architecture of the complex at low resolution, and combine this with high-resolution crystal structure determination of sub-complexes and individual proteins to achieve a quasi-atomic model for the full complex. In parallel, we will determine the ATPase, SUMO ligase and ubiquitin E3 ligase activities of the complex in the presence/absence of individual components (including Rad60), and with the inclusion of genetically characterised mutations in different components and domains for which the in vivo phenotype has already been defined. We will correlate these structural, biochemical and phenotypic data to define strong hypotheses for the roles of individual subunits and domains in the known functions of the Smc5/6 super-complex, and test these by epistasis and other genetic approaches in S.pombe. We will also use our structural and biochemical data in a reverse genetic approach to design targeted ?surgical? disruption of specific interfaces and interactions whose biochemical consequences can then be defined, with the aim of identifying separation-of-function mutations that affect only one of the biological processes to which Smc5/6 contributes.
These studies will provide profound insight into the structure, biochemistry and in vivo function of this important protein complex.
We wish to understand the in vivo functions of the Smc5/6 super-complex in terms of its biochemistry and its structure. In particular we wish to define the functional contributions of the individual subunits, determine how they assemble in the functional complex and sub-complexes, and elucidate the structural basis for regulation and coordination of the ATPase, SUMO ligase and ubiquitin E3 ligase activities, which the complex incorporates.
Towards this end we will develop expression and purification strategies for the assembled complex ex vivo from S.pombe, and the human and S.pombe complex, sub-complexes and individual proteins by recombinant expression in insect cells. We will use single-particle electron microscopy to define the overall architecture of the complex at low resolution, and combine this with high-resolution crystal structure determination of sub-complexes and individual proteins to achieve a quasi-atomic model for the full complex. In parallel, we will determine the ATPase, SUMO ligase and ubiquitin E3 ligase activities of the complex in the presence/absence of individual components (including Rad60), and with the inclusion of genetically characterised mutations in different components and domains for which the in vivo phenotype has already been defined. We will correlate these structural, biochemical and phenotypic data to define strong hypotheses for the roles of individual subunits and domains in the known functions of the Smc5/6 super-complex, and test these by epistasis and other genetic approaches in S.pombe. We will also use our structural and biochemical data in a reverse genetic approach to design targeted ?surgical? disruption of specific interfaces and interactions whose biochemical consequences can then be defined, with the aim of identifying separation-of-function mutations that affect only one of the biological processes to which Smc5/6 contributes.
These studies will provide profound insight into the structure, biochemistry and in vivo function of this important protein complex.
