Journal: DNA repair

Article Title: Screening of glycosylase activity on oxidative derivatives of methylcytosine: Pedobacter heparinus SMUG2 as a formylcytosine- and carboxylcytosine-DNA glycosylase.

doi: 10.1016/j.dnarep.2022.103408

Figure Lengend Snippet: Fig. 2. Screening of glycosylase activity on mC and its oxidative derivatives in UDG superfamily. GenBank acces sion numbers are shown after the species names. Family 1 (UNG, F1): Eco-UNG, Escherichia coli, NP_289138; Ngr- UNG1, Naegleria gruberi, XM_002679877.1; Ngr-UNG2, Naegleria gruberi, XM_002677827.1; Hsa-UNG, Homo sapi ens, NP_003353; Ipo-UNG, Ilyobacter polytropus DSM 2926, ADO84081.1; Nsa-UNG, Nitratifractor salsuginis strain E9I37–1 (DSM 176511), YP_004167197.1; Family 2 (MUG/ TDG, F2): Eco-MUG, Escherichia coli, P0A9H1; Hsa-TDG, Homo sapiens, NP_003202; Spo-TDG, Schizosaccharomyces pombe, O59825.1; Pma-MUG, Penicillium marneffei, NZ_A BAR01000000; Pla MUG, Planctomyces maris, WP_002644003.1. Family 3 (SMUG1/SMUG2/SMUG1-like, F3), Phe-SMUG2, Pedobacter heparinus, WP_012780920; Gme-SMUG1, Geobacter metallireducens GS-15, YP_383069; Lin-SMUG1-like, Listeria innocua, WP_010991469.1; Hsa- SMUG1a, Homo sapiens, NP_055126; Family 4 (UDGa, F4): Tth-UDGa, Thermus thermophilus HB27, AB109239.1; Msi- UDGa, Meiothermus Silvanus DSM 9946, YP_003685565.1; Rubrobacter xylanophilus (DSM) (Rxy), YP_643289.1. Fam ily 5 (UDGb, F5): Tth-UDGb, Thermus thermophilus HB27, YP_144415.1; Streptococcus mutans (Smu), WP_002263204.1. Family 6 (HDG, F6): Mba-HDG, Meth anosarcina barkeri str. Fusaro, YP_304295.1; Mac-HDG, Methanosarcina acetivoran, NP615428.1. The glycosylase reactions were performed at 37 ◦C for 1 h in a reaction mixture (10 μl) containing 1000 nM glycosylase protein, 10 nM oligodeoxynucleotide substrate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 mM DTT. Reactions were quenched by addition of 1 μl of 1 M NaOH. The reaction mixtures (2 μl) were mixed with 7.8 μl Hi-D formamide and 0.2 μl GeneScan 500 LI Size Standard and analyzed by Applied Biosystems 3130 × 1 genetic analyzer with a fragment analysis module. Cleavage products and remain ing substrates were quantified by GeneMapper software. A. U-containing substrate. B. hmU-containing substrate. C. fC- containing substrate. D. caC-containing substrate. All ex periments were performed in triplicate, and all data are expressed as the mean ± standard deviation.

Article Snippet: Some of the bacterial strains and DNA used in this study (Ilyobacter polytropus, Meiothermus silvanus, Rubrobacter xylanophilus) were purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Activity Assay, Software, Standard Deviation