2. Academic Validation
  3. Analysis of human cGAS activity and structure

Analysis of human cGAS activity and structure

  • Methods Enzymol. 2019:625:13-40. doi: 10.1016/bs.mie.2019.04.012.
Wen Zhou 1,  Aaron T Whiteley 1,  Philip J Kranzusch 2
Affiliations

Affiliations

  • 1 Department of Microbiology, Harvard Medical School, Boston, MA, United States; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, United States.
  • 2 Department of Microbiology, Harvard Medical School, Boston, MA, United States; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, United States; Parker Institute for Cancer Immunotherapy at Dana-Farber Cancer Institute, Boston, MA, United States. Electronic address: philip_kranzusch@dfci.harvard.edu.
PMID: 31455523 DOI: 10.1016/bs.mie.2019.04.012
Abstract

Cyclic GMP-AMP Synthase (cGAS) is an innate immune system enzyme responsible for recognition of double-stranded DNA aberrantly localized in the cell cytosol. cGAS binds DNA and is activated to catalyze production of the nucleotide second messenger 2'-5'/3'-5' cyclic GMP-AMP (2'3' cGAMP). In spite of a major role for cGAS in the cellular immune response, a complete understanding of cGAS biology has been limited by a lack of genetic tools to rapidly screen cGAS activity, instability of human cGAS-DNA interactions in vitro, and a previous absence of structural information for the human cGAS-DNA complex. Here we detail procedures to map the molecular determinants of cGAS activation and describe methods developed to prepare human cGAS-DNA crystals for structural analysis. Together with earlier systems established to study mammalian homologs of cGAS, these innovations provide a foundation to understand and therapeutically target human cGAS biology.

Keywords

Biochemistry; DNA sensing; Genetic screening; Innate immunity; Nucleotidyltransferase; STING; X-ray crystallography; cGAS.

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