l, RPA32-pEGFP-NLS foci MSD (Data shown as mean and weighted s
l, RPA32-pEGFP-NLS foci MSD (Data shown as mean and weighted s.e.m.; Resatorvid n=790 foci from 13 nuclei (CK-689), 823 foci from 12 nuclei (CK-666), 1135 foci from 13 nuclei (4-OHT). that nuclear actin-based mobility shapes chromatin organization by generating repair domains essential for HDR in eukaryotic cells. DSBs induce chromatin movement. In budding yeast, which repair DSBs primarily by HDR, induction of a single chromosomal break triggers increased local mobility: the DSB mean-square displacement is usually significantly higher than that of an undamaged region1,2. Moreover, multiple DSBs cluster after traversing long distances3. DSB clustering may facilitate homology search, increase repair efficiency or shield breaks from misrepair4,5. These movements are intricately related to HDR. Factors critical for resection initiation and downstream recombination are essential for DSB mobility in yeast1,2. In mammalian cells, DSBs are often described as more stable suggesting that NHEJ, the predominant repair pathway, limits movement6C8. However, in Resatorvid HeLa cells, Rad51-positive DSBs induced by alpha particles cluster4. Similarly, damaged telomeres in U2OS cells that are maintained by recombination merge in a Rad51-dependent manner9. Moreover, damaged active genes cluster in preparation for HDR5. Deprotected mouse telomere movements require the LINC complex which transmits cytoskeletal forces from the cytoplasm10. The molecular basis for DSB movement and DCHS2 its role in DNA repair, however, remain enigmatic. The machinery that drives actin polymerization in the cytoplasm is also found in the nucleus11. Specifically, the Arp2/3 complex as well as its activator WASP, a Wiskott-Aldrich syndrome family member, are located in both cellular compartments12C14. WASP brings the Arp2 and Arp3 subunits into close proximity to activate the complex and enable filament elongation15. Genotoxic agents Resatorvid trigger actin polymerization in the nucleoplasm of mammalian cells16; however, actin polymerizations role in DSB repair is not characterized. Actin nucleators bind damaged chromatin We performed an unbiased proteomics screen to document the recruitment of proteins to chromosomal DSBs in cell-free S-phase extracts derived Resatorvid from eggs. Peptides from control or DSB-containing chromatin protein fractions were labeled with isobaric tags and subjected to liquid chromatography mass spectrometry. We observed enrichment of known DSB repair regulators and proteins not previously associated with the DNA damage response (Extended Data Fig. 1a). Among such proteins were all seven subunits of the actin nucleating complex Arp2/3, as well as -actin and capping proteins (Extended Data Fig. 1a). We confirmed that -actin, Arpc4, and CapZ are recruited to Mre11-enriched, DSB-containing chromatin by Western blot (Fig. 1a). We next asked whether actin enrichment at chromosomal DSBs required DNA damage signaling. Inhibition of the PI3K-like kinases ATM and ATR reduced the binding of actin complexes to damaged chromatin (Extended Data Fig. 1b, c). Moreover, treatment with Resatorvid the small molecule inhibitor CK-666, which stabilizes the Arp2/3 complex in an open, inactive conformation17,18, decreased Arpc4, -actin, and CapZ enrichment in damaged chromatin (Fig. 1a, b). Overall, these results reveal that PI3K-like kinases and the Arp2/3 complex regulate the assembly of polymerized actin at chromosomal DSBs in extracts. Open in a separate window Physique 1 Actin complexes are recruited to damaged chromatina, Enrichment of actin complexes in damaged chromatin (+PflMI) by Western blot. Mre11 indicates DNA damage. b, Protein quantification in chromatin relative to +PflMI samples. (calculated by one-way ANOVA with multiple comparisons; data shown as mean and s.d.; n=5, 3, and 4 impartial experiments, left to right). WASP and Arp2/3 bind DSBs undergoing HDR We next tested whether WASP, an Arp2/3 activator, localized to DSB foci in mammalian.