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PMC:6145447 JSONTXT

Divisions (42) JSON TSV

div. section text # proj. length # Ann.
0 TIAB Nuclear Arp2/3 drives DNA break clustering for homology-directed repair DNA double-strand breaks (DS 1 1071 291 show
1 Body DSBs induce chromatin movement. In budding yeast, which repair DSBs primarily by HDR, induction of a 1 1728 0 show
2 Actin nucleators bind damaged chromatin We performed an unbiased proteomics screen to document the recruitment of proteins to chromosomal DS 1 1348 0 show
3 WASP and Arp2/3 bind DSBs undergoing HDR We next tested whether WASP, an Arp2/3 activator, localized to DSB foci in mammalian cells. DSB gene 1 2094 0 show
4 Arp2/3 inactivation impairs DSB movement The Arp2/3 complex generates propulsive forces by nucleating a highly-branched network of actin fila 1 4147 0 show
5 Arp2/3 nucleates actin foci at sites of HDR Genotoxic agents induce nuclear actin structures in mammalian cells16. In U2OS cells transfected wit 1 1446 0 show
6 WASP and Arp2/3 mediate HDR, not NHEJ To determine how DSB clustering impacts DNA repair pathways, we utilized a panel of U2OS cell lines 1 2307 0 show
7 Arp2/3 activity enhances DSB resection To assess the functional significance of Arp2/3-driven DSB mobility, we compared the repair kinetics 1 3022 0 show
8 Discussion DSB mobility is influenced by cell cycle stage, chromatin state, and repair pathway choice. Our find 1 1739 0 show
9 METHODS Chromatin binding assay and western blot Preparation of Low-Speed Supernatant (LSS) Xenopus egg extr 1 1806 0 show
10 METHODS Mass spectrometric analysis of DSB-containing chromatin Chromatin samples were isolated as described 1 1629 0 show
11 METHODS Cell culture and drug treatment U2OS and mouse-tail fibroblast cell lines were cultured in high-gluc 1 2576 0 show
12 METHODS Immunohistochemistry and quantification of DSB repair protein foci U2OS cells were cultured on 8-wel 1 3742 0 show
13 METHODS Chromatin immunoprecipitation ChIP experiments followed previously published protocols with the foll 1 2069 0 show
14 METHODS I-SceI-induced DSB repair assays U2OS cell lines which harbor chromosomally-integrated I-SceI-based 1 1522 0 show
15 METHODS Resection assay A detailed protocol for the resection assay has been recently described 37. Briefly, 1 2181 0 show
16 METHODS AsiSI-AID repair assay The repair assay followed recently published protocols with the following mod 1 1289 0 show
17 METHODS Live cell imaging U2OS cells stably expressing Rad52-mCherry, 53BP1-YFP, and Geminin-CFP constructs 1 1500 0 show
18 METHODS Nuclear actin-chromobody-tag-GFP imaging and analysis For live cell imaging experiments, U2OS cells 1 3019 0 show
19 METHODS DSB foci movement analysis DSB movement analysis followed the approach recently described 9. Nikon N 1 1881 0 show
20 METHODS DSB foci clustering analysis For analysis of DSB foci clustering during live cell imaging, a cluster 1 404 0 show
21 METHODS Quantification of RPA association to chromatin by flow cytometry Detection of chromatin-bound RPA in 1 1262 0 show
22 METHODS Clonogenic and cell survival assays U2OS cells were seeded onto 10 cm dishes (1000 cells per dish) o 1 1711 0 show
23 METHODS Quantification and Statistical Analysis Statistical parameters are reported in the Figures and the F 1 373 0 show
24 METHODS Research Animals The use of Xenopus laevis (African clawed frog) in this research proposal is approv 1 331 0 show
25 METHODS Data Availability The data that support the findings of this study are available from the correspond 1 281 0 show
27 Caption-Extended Data Fig. 1 Actin filament nucleators localize to chromosomal DSBs in Xenopus extracts and mammalian cells a, En 1 943 0 show
28 Caption-Extended Data Fig. 2 CK-689 does not significantly impact foci movement a, Summary of DSB movement analyses as related to 1 639 0 show
29 Caption-Extended Data Fig. 3 Arp2/3 clusters Rad51 foci a, Schematic of clustering events. b, Representative cell cycle distribut 1 424 0 show
30 Caption-Extended Data Fig. 4 WASP and Arp2/3 inactivation do not impair U2OS cell viability, nuclear area, nuclear sphericity or 1 1211 0 show
31 Caption-Extended Data Fig. 5 Arp2/3 enhances movement of 53BP1 foci in G2 a, Representative U2OS nuclei showing 53BP1-YFP foci tr 1 1186 0 show
32 Caption-Extended Data Fig. 6 Arp2/3 promotes actin foci assembly following DNA damage a, Representative U2OS nuclei showing class 1 1043 0 show
33 Caption-Extended Data Fig. 7 WASP and Arp2/3 mediate DSB repair by homology-directed mechanisms a, Representative FACS plots of G 1 1066 0 show
34 Caption-Extended Data Fig. 8 Actin nucleation regulates HDR in the nucleus and does not require formin-2 activity a, Western blot 1 940 0 show
35 Caption-Extended Data Fig. 9 B-lymphocytes derived from Wiskott-Aldrich Syndrome patients exhibit reduced DSB end-resection a, Re 1 1236 0 show
36 Caption-Extended Data Fig. 10 Arp2/3 inactivation confers sensitivity to DSBs induced in S-phase as well as replication stress-ind 1 1667 0 show
37 Caption-Figure 1 Actin complexes are recruited to damaged chromatin a, Enrichment of actin complexes in damaged chrom 1 367 0 show
38 Caption-Figure 2 Arp2/3 and WASP co-localize at HDR breaks a, Representative U2OS cells with WASP foci. b, Quantifica 1 785 0 show
39 Caption-Figure 3 Arp2/3 drives DSB mobility during HDR a, A representative U2OS nucleus with boxes indicating Rad52-m 1 1558 0 show
40 Caption-Figure 4 Nuclear actin foci cluster and localize to HDR sites a, Representative images of U2OS nuclei transfe 1 1269 0 show
41 Caption-Figure 5 WASP and Arp2/3 mediate HDR a, Summary of DR-GFP (HDR) assay (n=10, 6, 4, 4 left to right). ns, not 1 858 0 show
42 Caption-Figure 6 Arp2/3 facilitates resection and repair in G2 a, DSB repair at DSBs V-VI in G1-synchronized, ER-AsiS 1 1816 0 show