Arp2/3 and type-I myosins control chromosome mobility and end-resection at double-strand breaks in <em>S. cerevisiae</em>
收藏资源简介：
Using budding yeast, we show that the Arp2/3 actin branching complex has an evolutionarily conserved role in promoting chromosome mobility of double-strand breaks (DSBs). The radius of confinement of a broken chromosome is reduced by inhibiting Arp2/3 or by auxin-induced degron depletion of the nucleation-promoting factor Las17WASP or type-1 myosins. Arp2/3 and Las17 are required both to initiate and maintain 5 ' to 3' resection of DSB ends, whereas depleting Myo3 or Myo5 impairs broken chromosome motion without affecting resection. Conversely, inhibiting Exo1- and Dna2-dependent long-range resection reduces DSB mobility. Inactivating Arp2/3 before DSB induction leads to shortened checkpoint arrest, activating the Tel1ATM/Mre11 (TM) checkpoint. Shortened checkpoint arrest, but not reduced broken chromosome mobility per se, results in reduced interchromosomal homologous recombination. These results suggest that regulating the Arp2/3 complex plays a key role in the processing of DSB ends,..., Strain and plasmid construction All strains are derived from JKM179 (17), a well-characterized strain with the HML and HMR donor domains deleted. Ddc2 and Rad51 were GFP-tagged with a 13 amino acid linker GGSGGSRIPGLIN-eGFP as previously described (15). Ddc2-GGSGGSRIPGLIN-eGFP and Rad51-GGSGGSRIPGLIN-eGFP are referred to as Ddc2-GFP and Rad51-GFP, respectively, in this study. All AID-tagged mutant strains were derived from a modified version of JKM179 expressing osTIR1 at URA3 created by cutting pNHK53 (28) with StuI and integrating it into the genome. For degron-tagged derivatives, PCR products were generated with mixed oligos with homology to the C-terminal nd and plasmid pJH2892 or pJH2899 (27) to create a 9xMyc-AID (AID) insert with the KAN or NAT marker. Myo5-domain deletion plasmids (30) were digested with BamHI/SacI to excise a linear fragment for genome integration. Deletion of ORFs, myo5 mutants, and AID tags were introduced with the one-step ..., # Data from: Arp2/3 and type-I myosins control chromosome mobility and end-resection at double-strand breaks in *S. cerevisiae* Dataset DOI: [10.5061/dryad.f7m0cfz77](10.5061/dryad.f7m0cfz77) ## Description of the data and file structure **Date of Collection: **2020-2025 **Contributors: **Felix Y. Zhou, Marissa Ashton, Yiyang Jiang, Neha Arora, Kevin Clark, Kate B. Fitzpatrick, and James E. Haber **Overview** These datasets contain the mean squared displacement (MSD) analysis done to measure changes in double-strand break (DSB) and chromatin mobility in budding yeast in response to a single DSB. The MSD was measured using a custom MATLAB protocol that calculated the MSD curve and the radius of confinement (Rc). **Files and variables** **File:** MSD_and_Rc_-_Ddc2-GFP.xlsx File format: xlsx file Description: Data from MSD analysis of strains with a single Gal-HO induced DSB labeled with Ddc2-GFP. The spindle pole body (SPB) was labeled with Spc42-mCherry and ..., ,
本研究以出芽酵母为模型，证实Arp2/3肌动蛋白分支复合体（Arp2/3 actin branching complex）在促进双链断裂（double-strand breaks, DSBs）的染色体移动方面具有进化保守的功能。抑制Arp2/3，或通过生长素诱导的降解子（auxin-induced degron）系统耗竭成核促进因子（nucleation-promoting factor）Las17WASP或I型肌球蛋白，均可降低断裂染色体的约束半径（radius of confinement, Rc）。Arp2/3与Las17是启动并维持DSB末端5'→3'末端切除所需的核心因子；而耗竭Myo3或Myo5则会损伤断裂染色体的移动能力，但不影响末端切除过程。反之，抑制依赖Exo1与Dna2的长距离末端切除，则会降低DSB的移动性。在DSB诱导前使Arp2/3失活，会缩短检验点阻滞（checkpoint arrest）时长，进而激活Tel1ATM/Mre11（TM）检验点通路。检验点阻滞时长缩短（而非断裂染色体移动能力本身降低）会导致染色体间同源重组（homologous recombination）效率下降。上述结果表明，调控Arp2/3复合体在DSB末端加工过程中发挥关键作用…… 菌株与质粒构建 所有菌株均源自JKM179（17）——这是一株经过充分表征、缺失HML与HMR供体结构域的经典酵母菌株。如前文所述（15），我们通过含13个氨基酸接头GGSGGSRIPGLIN的eGFP对Ddc2与Rad51进行GFP标记，本研究中将Ddc2-GGSGGSRIPGLIN-eGFP与Rad51-GGSGGSRIPGLIN-eGFP分别简称为Ddc2-GFP与Rad51-GFP。所有AID标记的突变菌株均源自经过改造的JKM179菌株，该菌株通过将经StuI酶切的pNHK53（28）整合至基因组URA3位点以表达osTIR1。针对降解子标记的衍生菌株，我们使用与C端同源的混合寡核苷酸引物，结合质粒pJH2892或pJH2899（27），通过PCR扩增获得带有KAN或NAT筛选标记的9xMyc-AID（AID）插入片段。Myo5结构域缺失质粒（30）经BamHI/SacI双酶切后切下线性化片段用于基因组整合。ORF缺失、myo5突变体及AID标签的引入均采用一步法…… # 数据来自：《Arp2/3与I型肌球蛋白调控酿酒酵母（*S. cerevisiae*）双链断裂处的染色体移动与末端切除》（Arp2/3 and type-I myosins control chromosome mobility and end-resection at double-strand breaks in *S. cerevisiae*） 数据集DOI：10.5061/dryad.f7m0cfz77 ## 数据与文件结构说明 **采集时间：** 2020-2025 **贡献者：** Felix Y. Zhou、Marissa Ashton、Yiyang Jiang、Neha Arora、Kevin Clark、Kate B. Fitzpatrick及James E. Haber **概述** 本数据集包含用于检测出芽酵母中单个双链断裂诱导后双链断裂（DSB）与染色质移动变化的均方位移（mean squared displacement, MSD）分析数据。均方位移分析通过定制化MATLAB脚本完成，该脚本可计算MSD曲线与约束半径（Rc）。 **文件与变量说明** **文件：MSD_and_Rc_-_Ddc2-GFP.xlsx** 文件格式：XLSX文件 描述：该文件包含经Gal-HO诱导产生单个双链断裂、且带有Ddc2-GFP标记的菌株的MSD分析数据。实验中以Spc42-mCherry标记纺锤体极体（spindle pole body, SPB）……
