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 β-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.

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.

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