Study of IFI16 Protein Binding to DNA G-quadruplex Secondary Structure

abstrakt

angličtina

Interferon-gamma-induced protein 16 (IFI16) belongs to the highly homologous mammalian HIN-200 protein family. The IFI16 protein contains two tandemly located C-terminal DNA binding HIN domains and an N-terminal PYRIN domain. Both HIN domains show a highly conserved topology with a different surface charge distribution that affects their binding to DNA. The HIN domains of the IFI16 protein interact in vitro with both ssDNA and dsDNA through electrostatic interactions that are affected by salt concentrations, with the full-length IFI16 protein showing structure-specific binding to the G-quadruplex (G4) secondary structure. G4s are non-canonical local structures of DNA (or RNA) that readily form under physiological conditions in important regulatory regions of the genome or are part of the genomes of viruses and pathogens. In this study, the binding of the IFI16 protein to DNA was analyzed. We tested G4 sequence from Kaposi's sarcoma-associated herpesvirus (KSHV) and its mutant variants where one, two or three guanine tracks were replaced with adenine (KSHV-1no, KSHV-2no, and KSHV-3no). The biophysical characterization of the formation of secondary structures was tested by CD spectroscopy, the ThT assay, and electrophoretic mobility shift tests in a physiological environment without and with the addition of stabilizing potassium ions. The binding properties of the IFI16 protein to the formed DNA structures were studied in vitro (through binding and competitive binding EMSA) and in vivo (by functional assays in a yeast isogenic system). The results show that the analyzed sequences form G4 structures in vitro. However, compared to original KSHV derived sequence which form intramolecular G4s, the guanidine-track mutants formed mainly intermolecular complexes. In vitro binding and competitive binding experiments demonstrated specific binding of the IFI16 protein to G4 structures. Stabilization of G4 structures by IFI16 protein in vivo behind the p53 responsive element in the reporter gene promoter induced transcriptional repression of the given gene, while loss of G4-forming potential leads to increased p53 transactivation by its protein-protein interaction with IFI16. Our results point to the importance of structural features of DNA for exact gene regulation.

IFI16 protein; p53 protein, protein:DNA interaction; G-quadruplex; CD spectroscopy; EMSA; yeast one-hybrid isogenic system

KRATOCHVILOVÁ, L.; VALKOVÁ, N.; BRÁZDA, V.

30. 11. 2023

Vysoké učení technické v Brně, Fakulta chemická

Purkyňova 464/118, 612 00 Brno

978-80-214-6204-5

Studentská odborná konference Chemie je život 2023 Sborník abstraktů

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