Abstract
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.
Highlights
Barrier-to-autointegration factor, encoded by the BANF1 gene and here referred as BAF, is an abundant and ubiquitously expressed DNA binding protein with multiple functions important for maintaining the integrity of the cellular genome [1,2,3]
In order to identify the structural consequences of BAF phosphorylation by vaccinia-related kinase 1 (VRK1), we purified both a BAF construct in which the four cysteines are replaced by alanines, as reported in [29], and the catalytic domain of VRK1
The vaccinia-related kinases, belonging to the casein kinase family, phosphorylate BAF at Ser4, which is required for nuclear envelope disassembly and facilitates BAF release from chromatin [19,33,35]
Summary
Barrier-to-autointegration factor, encoded by the BANF1 gene and here referred as BAF, is an abundant and ubiquitously expressed DNA binding protein with multiple functions important for maintaining the integrity of the cellular genome [1,2,3]. BAF is highly conserved among metazoans, and BAF depletion is lethal during embryogenesis in Caenorhabditis elegans and Drosophila melanogaster [6,7,8] It was first identified as a host protein that captures viral DNA [1,9,10,11]. It is localized around the mitotic chromosomes [16], forming a dense cross-bridged chromatin that establishes a mechanical barrier and restricts nuclear membrane at the surface of chromatin It contributes to the sealing of the nuclear envelope by recruiting LEM-domain proteins [17] anchored at the inner nuclear membrane [18,19]. Cytoplasmic BAF localizes to the rupture site by binding to the leaking DNA, and recruits LEM-domain proteins anchored at the inner nuclear membrane, in order to trigger nuclear envelope repair and restore the nucleocytoplasmic barrier
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