The H2TH-like motif of the Escherichia coli multifunctional protein KsgA is required for DNA binding involved in DNA repair and the suppression of mutation frequencies

• Published in: Genes and environment Vol. 45; no. 1; p. 13
• Main Authors: Hayashi, Yuichiro, Funakoshi, Masafumi, Hirosawa, Kaname, Zhang-Akiyama, Qiu-Mei
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 12.04.2023; BioMed Central; BMC
• Subjects: 3D structure; Amino acids; Analysis; Base excision repair; Binding sites; C-Terminus; Cellular structure; Chimeras; Damage detection; Deoxyribonucleic acid; DNA; DNA binding; DNA glycosylase; DNA methylation; DNA repair; DNA-ligand interactions; E coli; Electrophoretic mobility; Enzymes; Escherichia coli; Gene deletion; Genes; Genetic aspects; Genetic research; Glycerol; H2TH domain; Hydrolases; In vivo methods and tests; Kasugamycin; Kinases; KsgA; Methyltransferase; Mutation; Oxidative stress; Plasmids; Protein binding; Proteins; Reactive oxygen species; RNA; rRNA; Structural analysis; Structure-function relationships; Vectors (Biology); Japan; H2TH domain; KsgA; 3D structure; DNA binding; Methyltransferase
• ISSN: 1880-7046; 1880-7062; 1880-7062
• DOI: 10.1186/s41021-023-00266-5

DNA oxidatively damaged by reactive oxygen species is repaired by base excision repair (BER) pathway proteins, with DNA glycosylases removing damaged or mismatched bases in the first step of BER. KsgA is a multifunctional protein that exhibits the activities of two enzymes, DNA glycosylase and rRNA dimethyltransferase. The structure-function relationship of the KsgA protein in cellular DNA repair remains unclear because the domains required for KsgA to recognize DNA have not been identified. To clarify the mechanisms by which KsgA recognizes damaged DNA and to identify the DNA-binding site, which exists in KsgA. A structural analysis and in vitro DNA-protein binding assay were performed. The C-terminal function of the KsgA protein was investigated in vitro and in vivo. The 3D conformations of KsgA, MutM, and Nei were compared at UCSF Chimera. The root mean square deviation of KsgA (214-273) and MutM (148-212) and that of KsgA (214-273) and Nei (145-212) were 1.067 and 1.188 Å, both less than 2 Å, suggesting that the C terminal of KsgA is spatially similar to the H2TH domains of MutM and Nei. The full-length KsgA protein and KsgA lacking 1-8 or 214-273 amino acids were purified and used in gel mobility shift assays. KsgA exhibited DNA-binding activity, which was lost in the C-terminally deleted KsgA protein. Spontaneous mutation frequency was measured using a mutM mutY ksgA-deficient strain, and the results obtained showed that the mutation frequency was not suppressed by KsgA lacking the C-terminal region, whereas it was in KsgA. To assess dimethyltransferase activity, kasugamycin sensitivity was assessed in wild-type and ksgA-deficient strains. Plasmids carrying the full-length ksgA gene and C-terminal deletion gene were introduced into ksgA-deficient strains. KsgA lacking the C terminus restored dimethyltransferase activity in the ksgA-deficient strain as well as KsgA. The present results confirmed that one enzyme exhibited two activities and revealed that the C-terminal (214-273) amino acids of KsgA were highly similar to the H2TH structural domain, exhibited DNA-binding activity, and inhibited spontaneous mutations. This site is not essential for dimethyltransferase activity.