Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

International audienceMCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nu-cleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 ˚ A resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure , but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 het-eromeric complex. Thermodynamic analysis of the quaternary complex together with structural model-ing support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway

Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction

International audienceThe histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-$T_{270}$ phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, $(R/K)R(I/A/V)$x$(L/P)$, the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-$F_{820}$ binds the same pocket of Asf1 as does histone $H4-F_{100}$. Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response

Conception sur une base rationnelle de peptides de haute affinité inhibant l'histone chaperon ASF1

International audienceAnti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.La fonction anti-silencing 1 (ASF1) est un chaperon d'histone H3-H4 conservé, impliqué dans la dynamique des histones pendant la réplication, la transcription et la réparation de l'ADN. Surexprimée dans les tissus en prolifération, y compris dans de nombreuses tumeurs, l'ASF1 est devenue une cible thérapeutique prometteuse. Ici, nous combinons des approches structurelles, informatiques et biochimiques pour concevoir des peptides qui inhibent l'interaction ASF1-histone. En partant de la structure du complexe ASF1-histone humain, nous avons mis au point une stratégie de conception rationnelle combinant la fixation des épitopes et l'optimisation des contacts d'interface pour identifier un puissant inhibiteur peptidique avec une constante de dissociation de 3 nM. Lorsqu'ils sont introduits dans des cellules en culture, les inhibiteurs entravent la prolifération cellulaire, perturbent la progression du cycle cellulaire et réduisent la migration et l'invasion des cellules d'une manière proportionnelle à leur affinité pour l'ASF1. Enfin, nous constatons que l'injection directe du plus puissant inhibiteur du peptide ASF1 dans les allogreffes de souris réduit la croissance des tumeurs. Nos résultats ouvrent de nouvelles voies pour utiliser les inhibiteurs de l'ASF1 comme des pistes prometteuses pour le traitement du cancer