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Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants. ispartof: BIOMOLECULES vol:10 issue:12 ispartof: location:Switzerland status: published

The nucleus is an organelle characteristic of eukaryotic cells and its mechanical properties play an essential role in the behavior of the cell, in particular its motility, polarity and survival. It is surrounded by an envelope comprising an inner membrane and an outer membrane, as well as a large number of proteins. These proteins are either anchored at the nuclear membrane, as emerin, or form a filament meshwork lining the inner nuclear membrane, as lamins. My thesis objectives were to understand molecular mechanisms deficient in two types of genetic diseases caused by mutations in inner nuclear envelope proteins: Emery-Dreifuss muscular dystrophy, associated to mutations in emerin and A-type lamins, and progeroid syndromes caused by mutations in A-type lamins. First, we showed that the emerin protein self- assembles in vitro and in cells (Herrada, Samson et al., ACS Chem. Biol., 2015). I then studied the structure of emerin oligomers, determined the minimal protein fragment necessary for the formation of these oligomers, identify residues forming the structural core of these oligomers by solid- state NMR in collaboration with the group of Prof A. Lange (FMP Berlin). And described the impact of emerin mutations causing Emery-Dreifuss muscular dystrophy on emerin self-assembly (Samson et al., Biomol. NMR Assign. 2016, Samson et al., FEBS J. 2017). Then, I observed, mainly using solution-state NMR, that only the self-assembled form of emerin is able to interact with A-type lamin tail, and that mutants causing Emery-Dreifuss muscular dystrophy and unable to self-assemble are also defective in A-type lamin binding. I also obtained preliminary data showing that phosphorylation of emerin by the Src kinase, observed after a mechanical stress in purified nuclei, regulates the interaction between self-assembled emerin and A-type lamins. Finally, I showed that the monomeric form of emerin is able to form a ternary complex with A-type lamin tail through the chromatin-associated protein Barrier-to- Autointegration Factor (BAF). After having measured the protein-protein affinities within this complex, identified the minimal protein fragments involved in the complex and developed a robust protocol for purification of this complex, I was able to obtain crystals under several conditions. Subsequently, I solved the 3D structure of this complex by molecular replacement at a resolution of 2 Å. Finally, I showed that mutations in A-type lamins causing autosomal recessive progeroid syndromes impair interaction with BAF in vitro, and our collaborators at Univ. Paris Diderot, the team of Dr B. Buendia, showed that these same mutations induce a significant decrease in the proximity between lamin A and BAF in HeLa cells. An article with me as a first author is in preparation that reports all these new data., Der Zellkern ist eine charakteristische Organelle einer eukaryotischen Zelle. Seine mechanischen Eigenschaften spielen eine wesentliche Rolle für das Verhalten der Zelle, insbesondere für deren Beweglichkeit, Polarität und Überleben. Es ist von einer Hülle umgeben, die eine innere Membran und eine äußere Membran sowie eine große Anzahl von Proteinen umfasst. Diese Proteine sind entweder an der Kernmembran verankert, als Emerin, oder bilden ein Filamentnetz, das die innere Kernmembran auskleidet, als Lamine. Ziel meiner Doktorarbeit war, molekulare Mechanismen zu verstehen, die bei zwei Arten genetischer Erkrankungen, die durch Mutationen in inneren Kernhüllproteinen verursacht werden, fehlen: Emery-Dreifuss-Muskeldystrophie, assoziiert mit Mutationen in Emerin- und A-Laminen, und Progeroid-Syndrome durch Mutationen in A- Laminen ein. Zunächst zeigten wir, dass das Emerin-Protein in vitro und in Zellen selbstorganisiert ist (Herada, Samson et al., ACS Chem. Biol., 2015). Ich untersuchte dann die Struktur von Emerin-Oligomeren, bestimmte das minimale Proteinfragment, das für die Bildung dieser Oligomere notwendig ist, identifiziere Reste, die den strukturellen Kern dieser Oligomere bilden, durch Festkörper-NMR in Zusammenarbeit mit der Gruppe von Prof. A. Lange (FMP Berlin) und beschrieb den Einfluss von Emerin-Mutationen, die Emery-Dreifuss- Muskeldystrophie auf die emerierende Selbstorganisation verursachen (Samson et al., Biomol. NMR Assign. 2016, Samson et al., FEBS J. 2017). Dann beobachtete ich, hauptsächlich unter Verwendung von Lösungs-NMR, dass nur die selbstorganisierte Form von Emerin in der Lage ist, mit dem A-Typ-Lamin- Schwanz zu interagieren, und dass Mutanten, die Emery-Dreifuss- Muskeldystrophie verursachen und nicht in der Lage sind, sich selbst zu assemblieren in A-Laminen. Ich erhielt auch vorläufige Daten, die zeigen, dass die Phosphorylierung von Emerin durch die Src-Kinase, die nach einer mechanischen Belastung in gereinigten Kernen beobachtet wird, die Wechselwirkung zwischen selbstorganisierten Emerin- und A-Typ-Laminen reguliert. Schließlich zeigte ich, dass die monomere Form von Emerin einen ternären Komplex mit dem A-Typ-Lamin-Tail durch das Chromatin-assoziierte Protein Barrier-to-Autointegration Factor (BAF) bilden kann. Nachdem ich die Protein-Protein-Affinitäten in diesem Komplex gemessen, die minimalen Proteinfragmente identifiziert und ein robustes Protokoll für die Reinigung dieses Komplexes entwickelt hatte, konnte ich Kristalle unter verschiedenen Bedingungen erhalten. Anschließend löste ich die 3D-Struktur dieses Komplexes durch molekularen Ersatz mit einer Auflösung von 2 Å. Schließlich zeigte ich, dass Mutationen in A-Laminen, die autosomal-rezessive Progeroid-Syndrome verursachen, die Interaktion mit BAF in vitro beeinträchtigen, und unsere Mitarbeiter von Univ. Paris Diderot, das Team von Dr. B. Buendia, zeigte, dass diese gleichen Mutationen eine signifikante Abnahme der Nähe zwischen Lamin A und BAF in HeLa-Zellen induzieren. Ein Artikel mit mir als Erstautor ist in Vorbereitung, der all diese neuen Daten berichtet.

Nucleic acids research / Special publication 49(7), 3841 - 3855 (2021). doi:10.1093/nar/gkab184, Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions., Published by Information Retrieval Ltd., London