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Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ?29 DNA mimic protein p56.

ABSTRACT: Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage 29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.

SUBMITTER: Banos-Sanz JI

PROVIDER: S-EPMC3711442 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage Φ29 DNA mimic protein p56.

Baños-Sanz José Ignacio JI Mojardín Laura L Sanz-Aparicio Julia J Lázaro José M JM Villar Laurentino L Serrano-Heras Gemma G González Beatriz B Salas Margarita M

Nucleic acids research 20130513 13

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage 29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the ...[more]

PMID: 23671337

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Project description:Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and T(15). Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12). All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation. The turning phosphate in the loop is located between T(13)and T(14). This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation. Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.

Dataset Information

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ?29 DNA mimic protein p56.

Publications

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage Φ29 DNA mimic protein p56.

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