RESUMEN
Adequate transport of large proteins that function in the nucleus is indispensable for cognate molecular events within this organelle. Selective protein import into the nucleus requires nuclear localization signals (NLS) that are recognized by importin receptors in the cytoplasm. Here we investigated the sequence requirements for nuclear targeting of Drosophila proteins involved in the metabolism of uracil-substituted DNA: the recently identified uracil-DNA degrading factor, dUTPase, and the two uracil-DNA glycosylases present in Drosophila. For the uracil-DNA degrading factor, NLS prediction identified two putative NLS sequences [PEKRKQE(320-326) and PKRKKKR(347-353)]. Truncation and site-directed mutagenesis using YFP reporter constructs showed that only one of these basic stretches is critically required for efficient nuclear localization in insect cells. This segment corresponds to the well-known prototypic NLS of SV40 T-antigen. An almost identical NLS segment is also present in the Drosophila thymine-DNA glycosylase, but no NLS elements were predicted in the single-strand-specific monofunctional uracil-DNA glycosylase homolog protein. This latter protein has a molecular mass of 31 kDa, which may allow NLS-independent transport. For Drosophila dUTPase, two isoforms with distinct features regarding molecular mass and subcellular distribution were recently described. In this study, we characterized the basic PAAKKMKID(10-18) segment of dUTPase, which has been predicted to be a putative NLS by in silico analysis. Deletion studies, using YFP reporter constructs expressed in insect cells, revealed the importance of the PAA(10-12) tripeptide and the ID(17-18) dipeptide, as well as the role of the PAAK(10-13) segment in nuclear localization of dUTPase. We constructed a structural model that shows the molecular basis of such recognition in three dimensions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Señales de Localización Nuclear/metabolismo , Pirofosfatasas/metabolismo , Uracilo/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Biología Computacional , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Humanos , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Estructura Cuaternaria de Proteína , Pirofosfatasas/química , Pirofosfatasas/genética , Alineación de Secuencia , SpodopteraRESUMEN
Uracil in DNA may arise by cytosine deamination or thymine replacement and is removed during DNA repair. Fruitfly larvae lack two repair enzymes, the major uracil-DNA glycosylase and dUTPase, and may accumulate uracil-DNA. We asked if larval tissues contain proteins that specifically recognize uracil-DNA. We show that the best hit of pull-down on uracil-DNA is the protein product of the Drosophila melanogaster gene CG18410. This protein binds to both uracil-DNA and normal DNA but degrades only uracil-DNA; it is termed Uracil-DNA Degrading Factor (UDE). The protein has detectable homology only to a group of sequences present in genomes of pupating insects. It is under detection level in the embryo, most of the larval stages and in the imago, but is strongly upregulated right before pupation. In Schneider 2 cells, UDE mRNA is upregulated by ecdysone. UDE represents a new class of proteins that process uracil-DNA with potential involvement in metamorphosis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Uracilo/metabolismo , Secuencia de Aminoácidos , Animales , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia MolecularRESUMEN
The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical beta-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged.