Analysis of human cGAS activity and structure

Methods Enzymol. 2019:625:13-40. doi: 10.1016/bs.mie.2019.04.012. Epub 2019 May 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • DNA / chemistry
  • DNA / metabolism
  • Humans
  • Immunity, Innate / physiology
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / metabolism*
  • Signal Transduction / physiology

Substances

  • Membrane Proteins
  • STING1 protein, human
  • DNA
  • Nucleotidyltransferases
  • cGAS protein, human