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.
|Bhat, Kamakoti P; Krishnamoorthy, Archana; Dungrawala, Huzefa et al. (2018) RADX Modulates RAD51 Activity to Control Replication Fork Protection. Cell Rep 24:538-545|
|Sallmyr, Annahita; Tomkinson, Alan E (2018) Repair of DNA double-strand breaks by mammalian alternative end-joining pathways. J Biol Chem 293:10536-10546|
|Warren, Garrett M; Stein, Richard A; Mchaourab, Hassane S et al. (2018) Movement of the RecG Motor Domain upon DNA Binding Is Required for Efficient Fork Reversal. Int J Mol Sci 19:|
|Moiani, Davide; Ronato, Daryl A; Brosey, Chris A et al. (2018) Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities. Methods Enzymol 601:205-241|
|Polyzos, Aris A; Wood, Nigel I; Williams, Paul et al. (2018) XJB-5-131-mediated improvement in physiology and behaviour of the R6/2 mouse model of Huntington's disease is age- and sex- dependent. PLoS One 13:e0194580|
|Schneidman-Duhovny, Dina; Hammel, Michal (2018) Modeling Structure and Dynamics of Protein Complexes with SAXS Profiles. Methods Mol Biol 1764:449-473|
|Sung, Patrick (2018) Introduction to the Thematic Minireview Series: DNA double-strand break repair and pathway choice. J Biol Chem 293:10500-10501|
|Shen, Jianfeng; Ju, Zhenlin; Zhao, Wei et al. (2018) ARID1A deficiency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade. Nat Med 24:556-562|
|Sengupta, Shiladitya; Yang, Chunying; Hegde, Muralidhar L et al. (2018) Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress. DNA Repair (Amst) 66-67:1-10|
|Mu, Hong; Geacintov, Nicholas E; Broyde, Suse et al. (2018) Molecular basis for damage recognition and verification by XPC-RAD23B and TFIIH in nucleotide excision repair. DNA Repair (Amst) :|
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