Direct recruitment of TONSOKU by the N-terminal tails of histone variants H2A.X and H2A.W coordinates DNA repair

Histone variants play crucial roles in DNA replication and DNA damage repair. Plant TONSOKU (TSK) specifically recognizes unmethylated histone H3.1 to bind post-replicative chromatin for DNA repair, but the precise mechanisms of TSK recruitment to double-stranded break (DSB) sites remain unclear. Here, we discovered that the leucine-rich repeats (LRR) domain of TSK recognizes the N-terminal tail of the histone variant H2A.X, the phosphorylated form of which (also known as ?-H2A.X) is a critical marker of DSBs at both replication forks and euchromatin. Additionally, the LRR domain of TSK recognizes the N-terminal tail of the plant-specific histone H2A variant H2A.W, which is essential for DSB repair in heterochromatin, but not H2A.Z, which primarily regulates gene expression. A motif containing multiple basic and acidic residues (BAR motif), unique to H2A.Z, prevents TSK from binding. Notably, mutations that abolish TSK-H2A.X/W binding interfere with TSK-mediated DNA repair and root development. Genetic analyses indicate that H2A.X and H2A.W are redundantly required for DNA damage repair in vivo, with the N-terminus of H2A.X being particularly indispensable. This study reveals a novel H2A.X/W-dependent mechanism for recruiting the DNA repair protein TSK, highlighting the critical role of their previously uncharacterized N-terminal tails in plants. Overall design: RNA-seq profiling of Col, h2a.x-1, h2a.w-2, tsk and h2a.x/w-2 in 7-day-old seedling