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Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage
Molecular Biology of the Cell ( IF 3.1 ) Pub Date : 2021-08-11 , DOI: 10.1091/mbc.e20-10-0680
Verena Hurst 1, 2 , Kiran Challa 1 , Kenji Shimada 1 , Susan M Gasser 1, 2
Affiliation  

Upon induction of DNA damage with 405 nm laser light, proteins involved in Base Excision Repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by Latrunculin B, Cytochalasin B or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by Nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin, but also of tubulin. Our study is one of few linking cytoskeletal integrity to BER.

Movie S1: 405 nm-laser damage does not trigger actin recruitment. The GFP2-tagged nuclear Actin-Chromobody (Chromotek), which binds both F- and G-actin does not respond to 405 nm laser-induced DNA damage. U2OS cells expressing the nuclear actin chromobody from a plasmid at high (left) or low level (right) were exposed to laser damage and imaged over time.Download Original Video (1.0 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-10-0680/20210810/media/mc-e20-10-0680-s03.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c394aa1c604dd45b92d24b41b0bc3db9e24902f34e617cd406d643b182a4612be08b0ed0e18c1b6dac88b46768b4b79514d789ee756677b0c8a4d48986fb5685c11e249501e0468911aa0da330998396cadffb31f911666Movie S2: Nocodazole leads to prolonged PCNA accumulation at laser-induced damage. U2OS cells expressing GFP-PCNA were treated with DMSO (left) or 330 nM Nocodazole (right) for 30 min prior to induction of photodamage with a 405 nm laser. Images were acquired over time as described in the methods section.Download Original Video (.8 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-10-0680/20210810/media/mc-e20-10-0680-s04.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c394aa1c604dd45b92d24b41b0bc3db9e24902f34e617cd406d643b182a4612be08b0ed0e18c1b6dac88b41768b4b79514d78134f50b197c63a94699e1e8953a1800d24a09980287b334ba527f7fa072a29594943d95128


中文翻译:

细胞骨架完整性影响 DNA 损伤时的 XRCC1 和 PCNA 动力学

在用 405 nm 激光诱导 DNA 损伤后,参与碱基切除修复 (BER) 的蛋白质被募集到 DNA 损伤中。我们发现,短补丁 (XRCC1) 或长补丁 (PCNA) BER 的典型因子动态会被干扰人体细胞中肌动蛋白或微管蛋白聚合的化学物质改变。Latrunculin B、Cytochalasin B 或 Jasplakinolide 对肌动蛋白丝的不稳定会降低激光诱导损伤时的 BER 因子积累,而 Nocodazole 对微管蛋白聚合的抑制会增加它。我们没有检测到肌动蛋白募集到激光诱导的 DNA 损伤部位,但细胞质肌动蛋白丝的解聚提高了细胞核中的肌动蛋白和微管蛋白信号。虽然已发表的证据表明 F-肌动蛋白在哺乳动物的双链断裂修复中具有积极作用,出芽酵母细胞核中肌动蛋白的富集会干扰 BER,从而增加对 Zeocin 的敏感性。我们的定量成像结果表明,细胞质肌动蛋白的解聚可能会损害哺乳动物的 BER 效率,这不仅是由于核肌动蛋白水平升高,还因为微管蛋白水平升高。我们的研究是将细胞骨架完整性与 BER 联系起来的少数研究之一。

电影 S1: 405 nm 激光损伤不会触发肌动蛋白募集。结合 F-肌动蛋白和 G-肌动蛋白的 GFP2 标记的核肌动蛋白染色体 (Chromotek) 对 405 nm 激光诱导的 DNA 损伤没有反应。U2OS 细胞以高(左)或低(右)的水平从质粒表达核肌动蛋白染色体,暴露在激光损伤下并随着时间的推移成像。下载原始视频 (1.0 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-10-0680/20210810/media/mc-e20-10-0680-s03.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c394aa1c604dd45b92d24b41b0bc3db9e24902f34e617cd406d643b182a4612be08b0ed0e18c1b6dac88b46768b4b79514d789ee756677b0c8a4d48986fb5685c11e249501e0468911aa0da330998396cadffb31f911666电影 S2:诺考达唑导致激光诱导损伤时 PCNA 积累时间延长。在用 405 nm 激光诱导光损伤之前,用 DMSO(左)或 330 nM Nocodazole(右)处理表达 GFP-PCNA 的 U2OS 细胞 30 分钟。如方法部分所述,随着时间的推移获取图像。下载原始视频 (.8 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-10-0680/20210810/media/mc-e20-10-0680-s04.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c394aa1c604dd45b92d24b41b0bc3db9e24902f34e617cd406d643b182a4612be08b0ed0e18c1b6dac88b41768b4b79514d78134f50b197c63a94699e1e8953a1800d24a09980287b334ba527f7fa072a29594943d95128
更新日期:2021-08-12
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