Characterization of Bacillus subtilis uracil-DNA glycosylase and its inhibition by phage [straight phi]29 protein p56

• Published in: Molecular microbiology 2011-06, Vol.80 (6), p.1657
• Main Authors: Pérez-Lago, Laura, Serrano-Heras, Gemma, Baños, Benito, Lázaro, José M, Alcorlo, Martín, Villar, Laurentino, Salas, Margarita
• Format: Article
• Language: English
• Subjects: Bacterial proteins; Biochemistry; DNA repair; Enzymes; Gram-positive bacteria
• ISSN: 0950-382X; 1365-2958

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 ...29 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 ...29 genome. In this work, we also show that a ...29-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 ...29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition. (ProQuest: ... denotes formulae/symbols omitted.)