Cellular processes are influenced by liquid phase separation, but its role in DNA repair is unclear. Here, we show that in Saccharomyces cerevisiae, liquid droplets made up of DNA repair proteins cooperate with different types of DNA damage-inducible intranuclear microtubule filaments (DIMs) to promote the clustering of DNA damage sites and maintain genome stability. Rad52 DNA repair proteins at different DNA damage sites assemble in liquid droplets that fuse into a repair centre droplet via the action of petite DIMs (pti-DIMs). This larger droplet concentrates tubulin and projects short aster-like DIMs (aster-DIMs), which tether the repair centre to longer DIMs mediating the mobilization of damaged DNA to the nuclear periphery for repair. Our findings indicate that cooperation between Rad52 liquid droplets and various types of nuclear filaments promotes the assembly and function of the DNA repair centre.

Oshidari et al.2 Cellular processes are influenced by liquid phase separation, but its role in DNA repair is unclear. Here, we show that in Saccharomyces cerevisiae, Rad52 DNA repair proteins at different DNA damage sites assemble liquid droplets that fuse into a repair centre droplet. This larger droplet concentrates tubulin and projects short aster-like microtubule filaments, which tether the droplet to longer microtubule filaments mediating the mobilization of damaged DNA to the nuclear periphery for repair. Eukaryotic genomes are dynamic structures and are non-randomly arranged within the cell nucleus, which is defined by an envelope perforated with nuclear pore complexes (NPCs) 1 . Genome dynamics allow cells to repair DNA double-strand breaks (DSBs), which are highly toxic DNA lesions that trigger the DNA damage checkpoint 2 . Specifically, the movement of DSBs allows them to escape repair-repressive heterochromatin domains, search for homologous sequences or localize to repair-conducive NPCs 3-15 .The de novo assembly of intranuclear filaments, onto which DSBs are transported by motor proteins, promotes DSB escape from heterochromatin or movement to NPCs 6,16-18 . In S. cerevisiae cells with a single DSB, the Kinesin-14 motor proteins Kar3 and Cik1 associate with the break site and are required for its capture by long DNA damage-inducible intranuclear microtubule filaments (DIMs), which emanate from the microtubule-organizing centre (MTOC) 6,16 . The break is then directionally mobilized by Kinesin-14 onto a DIM and moved away from the MTOC to NPCs for repair 16 . Similarly, in cells treated with carcinogens such as methyl methanesulfonate (MMS), damaged DNA, identified by the presence of the Rad52 DNA repair protein, moves along DIMs to NPCs, where the focus later dissolves, marking repair completion 16 . In flies, a similar actin/myosin-based mechanism moves DSBs for repair 8,18 . Importantly, in a given cell, carcinogens can trigger several DSBs that co-localize and create a DNA repair centre, which is enriched in Rad52 in yeast but remains poorly understood across eukaryotes 17,19 . The forces driving DSB clustering, whether such forces crosstalk with nuclear filaments, and how clustering promotes genome stability remain unclear.Therefore, we used a yeast system for the fluorescence-based visualization of DSBindicating Rad52, a-tubulin Tub1, and NPC-indicating Nup49 protein 16 ( Supplementary Fig. 1ab). Cells treated with MMS exhibited Rad52/RPA-positive DSBs ( Supplementary Fig. 1c). MMS induced one DIM in cells containing a single large and bright Rad52 focus ( Fig. 1a-b). DIMs emanated from the MTOC and efficiently captured the large Rad52 focus, as expected (Fig. 1c) 16 . In contrast, the MTOC of cells containing more than one Rad52 focus tended to exhibit several shorter microtubule filaments (denoted petite DIMs or pti-DIMs) that failed to capture damaged DNA (Fig. 1a-c). Thus, cells with several DSB-indicating Rad52 foci exhibit several pti-DIMs, which, in contrast to the DIM in cells with one ...

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