title: Characterization of Bacillus subtilis uracil-DNA glycosylase and its inhibition by phage φ29 protein p56.
abstract: Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp-65 and His-187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe-78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29-related phage, GA-1, encodes a p56-like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe-191 of B. subtilis UDG is critical for the interaction with φ29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.
authors.affiliation: Instituto de Biología Molecular Eladio Viñuela, CSIC, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Pérez-Lago
authors.forename: Gemma
authors.initials: G
authors.lastname: Serrano-Heras
authors.forename: Benito
authors.initials: B
authors.lastname: Baños
authors.forename: José M
authors.initials: JM
authors.lastname: Lázaro
authors.forename: Martín
authors.initials: M
authors.lastname: Alcorlo
authors.forename: Laurentino
authors.initials: L
authors.lastname: Villar
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D014764:Viral Proteins; D051981:Uracil-DNA Glycosidase