Spotlight: DNA Resource Core
June 29, 2018
In this DF/HCC eNews Spotlight Edition, we present the DF/HCC DNA Resource Core, which provides access to specialized resources such as rapid and user-friendly Sanger DNA sequencing services and plasmid clone repository and distribution services. Founded in 1999 and established as a CCSG Core in 2000, the Core addresses both small- and large-scale DNA sequencing and plasmid-related needs.
The DNA Resource Core joins the Collaborative Functional Genomics and Transgenic Mouse Core in participating in a CRISPR core program with Sigma Millipore that provides members with access to expert advice and discounts on reagents for CRISPR technologies.
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- DNA Sequencing (Sanger sequencing technology)
- Plasmid Clones (empty vectors, cDNA clone collections, and more)
- The PlasmID Repository (deposit clones for distribution to the community)
Development of new technology for the study of membrane proteins, PROTEIN COMPLEXES, AND VIRUSES
PIs: G. WagnerHMS(Cancer Cell Biology Program)
Detailed analyses of membrane proteins, large protein complexes, and viruses can contribute to the ability to understand the roles of proteins and viruses in cancer cell biology. In this study, Wagner and colleagues improved the current state-of-the-art in nanodiscs used for nuclear magnetic resonance (NMR) by engineering covalently circularized nanodiscs. They then applied the technology to the visualization of a membrane protein and of entry of a non-envelope virus into cells. The technical advance they report has application for both NMR and cryo-electron microscopy (cryo-EM) studies. The report includes a detailed protocol and the plasmid vectors described in the study were of immediate interest to a broad group of researchers.
Contribution of the Core: The Core added plasmid vectors reported in this study to the PlasmID repository, making them broadly available to the research community and alleviating the burden of plasmid distribution from the Wagner lab.
Publication: Nasr ML, Baptista D, Strauss M, Sun ZJ, Grigoriu S, Huser S, Plückthun A, Hagn F, Walz T, Hogle JM, Wagner G. Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods. 2017 Jan;14(1):49-52. PMCID: PMC5199620.
Development of a large-scale community resource for in vivo study of cancer cell biology and other topics.
PI: N. PerrimonHMS(Cancer Cell Biology, Cancer Genetics Programs)
Studies in human cell lines have tremendous value but can be meaningfully supplemented by in vivo studies that address questions related to cancer cell biology, metastasis, cachexia, and other topics. Research using fruit fly Drosophila melanogaster has contributed a wealth of understanding relevant to cancer biology and treatments, including discovery of core components of many signal transduction pathways. In this work, Perrimon and colleagues developed a large-scale resource of fly stocks useful for in vivo RNAi studies, including studies in which genes are knocked down in specific stages and/or tissues. The resource is publicly available and opens the doors wide to new studies of cancer biology, other human diseases, and other topics in this exemplary in vivo genetic model system. The fly stocks reported by Perrimon and colleagues have been distributed to more than 2,500 laboratories.
Contribution of the Core: The core provided Sanger DNA sequencing services that were essential for quality analysis of the plasmids and transgenic fly stocks generated in this large-scale resource production effort. The core also distributes plasmid vectors published in this study through the PlasmID repository.
Publication: Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, Yang-Zhou D, Flockhart I, Binari R, Shim HS, Miller A, Housden A, Foos M, Randkelv S, Kelley C, Namgyal P, Villalta C, Liu LP, Jiang X, Huan-Huan Q, Wang X, Fujiyama A, Toyoda A, Ayers K, Blum A, Czech B, Neumuller R, Yan D, Cavallaro A, Hibbard K, Hall D, Cooley L, Hannon GJ, Lehmann R, Parks A, Mohr SE, Ueda R, Kondo S, Ni JQ, Perrimon NHMS. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics. 2015 Nov;201(3):843-52. PMCID: PMC4649654.
Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis.
PIs: A. D’AndreaDFCI(Breast Cancer, Cancer Cell Biology Programs)
Timely DNA replication across damaged DNA is critical for maintaining genomic integrity. Translesion DNA synthesis (TLS) allows bypass of DNA lesions using error-prone TLS polymerases. The E3 ligase RAD18 is necessary for proliferating cell nuclear antigen (PCNA) monoubiquitination and TLS polymerase recruitment; however, the regulatory steps upstream of RAD18 activation are less understood. Here, the D’Andrea lab showed that the UBZ4 domain-containing transcriptional repressor ZBTB1 is a critical upstream regulator of TLS. This study was facilitated by the use of ZBTB1 clones provided through the DNA Resource Core. The UBZ4 motif is required for PCNA monoubiquitination and survival after UV damage. ZBTB1 associates with KAP-1, a transcriptional repressor whose phosphorylation relaxes chromatin after DNA damage. ZBTB1 depletion impairs formation of phospho-KAP-1 at UV damage sites and reduces RAD18 recruitment. Furthermore, phosphorylation of KAP-1 is necessary for efficient PCNA modification. We propose that ZBTB1 is required for localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair.
Contribution of the Core: The core provided access through the core’s PlasmID repository to the ZBTB1 and DVC1 open reading frame clones that were used in this study.
Publication: Kim HDFCI, Dejsuphong D, Adelmant G, Ceccaldi R, Yang K, Marto JADFCI, D'Andrea ADDFCI. Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis. Mol Cell. 2014 Apr 10;54(1):107-18. PMC3989074.
For more information or to contact the DNA Resource Core, visit the core website here.