Core 1 ? Expression and Molecular Biology (EMB) Core PROJECT SUMMARY/ABSTRACT The overall goal of SBDR-4 is to characterize the DNA repair multi-protein machines and assemblies that maintain genomic integrity and to identify the mechanisms that underlie detection and signaling of the damage, in order to recruit repair machines to the damage site for repair. The proposed SBDR research will provide mechanistic, predictive biology for cancer interventions through a framework built upon structural and functional understanding of DNA repair machines. The major challenges faced by SBDR-4 include: (1) efficient reconstitution and assembly of full-length and modified proteins and complexes that control integrity of the genome, (2) determining the solution structures of flexible multi-protein complexes that are spatially and temporally regulated in cells, and (3) linking structures to biochemistry and cellular phenotypes. The Expression and Molecular Biology (EMB) Core will overcome the technical aspects of these bottlenecks by implementing robust DNA assembly technologies, incorporating efficient protein purification strategies, and developing advanced tools for cellular studies. The EMB Core services will empower SBDR-4 by providing high-quality key starting materials for cell biological, biochemical, biophysical, and structural investigations of DNA repair machines. The EMB Core will serve as a research, production, and development resource for all five Projects by providing dedicated Core staff and centralized expertise. The EMB Core will develop robust pipelines for (1) construction of single-gene and multi-gene expression vectors, (2) creation of isogenic human cell lines with designed changes, (3) streamlined scaled-up expression technologies, (4) purification of well- behaved, functional DNA repair proteins and complexes, and (5) validation of protein interactions and partnerships. The innovative technologies provided by EMB Core to enable SBDR-4 research include: (1) high- efficiency cloning of multi-gene vectors using advanced DNA assembly technologies, e.g. the BioBrick-based MacroBac system, Gibson assembly and Golden Gate assembly; (2) incorporation of cleavable GFP tags and application of GFP-binder single-chain nanobody columns for efficient purification of multimeric protein complexes for in vitro analysis; and (3) developing novel cellular tools using a novel, highly effective CRISPR knock-in (KI) method to create isogenic human cell lines with desired changes, as well as using the HIV-TAT methodology to produce interfering peptides for cellular studies. The EMB Core thus offers a robust blend of established and new technologies and efficiently provides reagents to jump-start Project Aims and cross- Project interactions for characterizing transient interactions and dynamic conformations that control the assembly and function of multi-protein complexes in response to DNA damage to maintain genomic stability.
Core 1 ? Expression and Molecular Biology (EMB) Core PORJECT NARRATIVE The proposed research and production services and new technology platforms developed by the Expression and Molecular Biology (EMB) Core will provide centralized and critical resources for the SBDR Program and create significant synergy with all five Projects. We will accelerate the pace of proposed SBDR research on the DNA Repair machines that serve to maintain genomic integrity to achieve the SBDR goal of mechanistic, predictive biology for improved cancer interventions that will be highly relevant to public health.
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|Tsai, Chi-Lin; Tainer, John A (2018) Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes. Methods Enzymol 599:157-196|
|Ogorzalek, Tadeusz L; Hura, Greg L; Belsom, Adam et al. (2018) Small angle X-ray scattering and cross-linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy. Proteins 86 Suppl 1:202-214|
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