Ancient genomes reveal origin and rapid trans-Eurasian migration of 7th century Avar elites.

The Avars settled the Carpathian Basin in 567/68 CE, establishing an empire lasting over 200 years. Who they were and where they came from is highly debated. Contemporaries have disagreed about whether they were, as they claimed, the direct successors of the Mongolian Steppe Rouran empire that was destroyed by the Turks in ∼550 CE. Here, we analyze new genome-wide data from 66 pre-Avar and Avar-period Carpathian Basin individuals, including the 8 richest Avar-period burials and further elite sites from Avar's empire core region. Our results provide support for a rapid long-distance trans-Eurasian migration of Avar-period elites. These individuals carried Northeast Asian ancestry matching the profile of preceding Mongolian Steppe populations, particularly a genome available from the Rouran period. Some of the later elite individuals carried an additional non-local ancestry component broadly matching the steppe, which could point to a later migration or reflect greater genetic diversity within the initial migrant population.

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition.

Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition.

The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase.

DNA metabolism and repair is vital for the maintenance of genome integrity. Specific proteinaceous inhibitors of key factors in this process have high potential for deciphering pathways of DNA metabolism and repair. The dUTPase enzyme family is responsible for guarding against erroneous uracil incorporation into DNA. Here, we investigate whether the staphylococcal Stl repressor may interact with not only bacterial but also eukaryotic dUTPase. We provide experimental evidence for the formation of a strong complex between Stl and Drosophila melanogaster dUTPase. We also find that dUTPase activity is strongly diminished in this complex. Our results suggest that the dUTPase protein sequences involved in binding to Stl are at least partially conserved through evolution from bacteria to eukaryotes.

Potential steps in the evolution of a fused trimeric all-ß dUTPase involve a catalytically competent fused dimeric intermediate.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer-like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two-repeat-containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three-fold structural symmetry.

NLS copy-number variation governs efficiency of nuclear import--case study on dUTPases.

Nucleocytoplasmic trafficking of large macromolecules requires an active transport machinery. In many cases, this is initiated by binding of the nuclear localization signal (NLS) peptide of cargo proteins to importin-α molecules. Fine orchestration of nucleocytoplasmic trafficking is of particularly high importance for proteins involved in maintenance of genome integrity, such as dUTPases, which are responsible for prevention of uracil incorporation into the genome. In most eukaryotes, dUTPases have two homotrimeric isoforms: one of these contains three NLSs and is present in the cell nucleus, while the other is located in the cytoplasm or the mitochondria. Here we focus on the unusual occurrence of a pseudo-heterotrimeric dUTPase in Drosophila virilis that contains one NLS, and investigate its localization pattern compared to the homotrimeric dUTPase isoforms of Drosophila melanogaster. Although the interaction of individual NLSs with importin-α has been well characterized, the question of how multiple NLSs of oligomeric cargo proteins affect their trafficking has been less frequently addressed in adequate detail. Using the D. virilis dUTPase as a fully relevant physiologically occurring model protein, we show that NLS copy number influences the efficiency of nuclear import in both insect and mammalian cell lines, as well as in D. melanogaster and D. virilis tissues. Biophysical data indicate that NLS copy number determines the stoichiometry of complexation between importin-α and dUTPases. The main conclusion of our study is that, in D. virilis, a single dUTPase isoform efficiently reproduces the cellular dUTPase distribution pattern that requires two isoforms in D. melanogaster.