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Physiological truncation and domain organization of a novel uracil‐DNA‐degrading factor
Author(s) -
Pukáncsik Mária,
Békési Angéla,
Klement Éva,
HunyadiGulyás Éva,
Medzihradszky Katalin F.,
Kosinski Jan,
Bujnicki Janusz M.,
Alfonso Carlos,
Rivas Germán,
Vértessy Beáta G.
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07556.x
Subject(s) - uracil , dna , drosophila melanogaster , biology , dna binding domain , dna binding site , biochemistry , transcription factor , genetics , microbiology and biotechnology , gene , promoter , gene expression
Uracil in DNA is usually considered to be an error, but it may be used for signaling in Drosophila development via recognition by a novel uracil‐DNA‐degrading factor (UDE) [(Bekesi A et al. (2007) Biochem Biophys Res Commun 355 , 643–648]. The UDE protein has no detectable similarity to any other uracil‐DNA‐binding factors, and has no structurally or functionally described homologs. Here, a combination of theoretical and experimental analyses reveals the domain organization and DNA‐binding pattern of UDE. Sequence alignments and limited proteolysis with different proteases show extensive protection by DNA at the N‐terminal duplicated conserved motif 1A/1B segment, and a well‐folded domain within the C‐terminal half encompassing conserved motifs 2–4. Theoretical structure prediction suggests that motifs 1A and 1B fold as similar α‐helical bundles, and reveals two conserved positively charged surface patches that may bind DNA. CD spectroscopy also supports the presence of α‐helices in UDE. Full functionality of a physiologically occurring truncated isoform in Tribolium castaneum lacking one copy of the N‐terminal conserved motif 1 is revealed by activity assays of a representative truncated construct of Drosophila melanogaster UDE. Gel filtration and analytical ultracentrifugation results, together with analysis of predicted structural models, suggest a possible dimerization mechanism for preserving functionality of the truncated isoform. Structured digital abstract• MINT‐7385914 : UDE (uniprotkb: Q961C4 ) and UDE (uniprotkb: Q961C4 ) bind ( MI:0407 ) by cosedimentation in solution ( MI:0028 )