Physiological truncation and domain organization of a novel uracil-DNA-degrading factor.
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 alpha-helical bundles, and reveals two conserved positively charged surface patches that may bind DNA. CD spectroscopy also supports the presence of alpha-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.
Pukáncsik
Mária
M
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
Békési
Angéla
A
Klement
Eva
E
Hunyadi-Gulyás
Eva
E
Medzihradszky
Katalin F
KF
Kosinski
Jan
J
Bujnicki
Janusz M
JM
Alfonso
Carlos
C
Rivas
Germán
G
Vértessy
Beáta G
BG
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002942:Circular Dichroism; D019295:Computational Biology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D020033:Protein Isoforms / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017384:Sequence Deletion
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D020033:Protein Isoforms; C519400:UDE protein, Drosophila; D004247:DNA