Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks.
Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks.
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DOI：
10.1038/s41467-022-32743-w
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发表时间：
2022-09-08
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
DNA双链断裂（DSB）通过多种不同的途径修复，结果从无错误修复到诱变和基因组丢失。DSB修复途径的串扰和补偿尚未完全理解，尽管它对基因组稳定性，肿瘤发生和使用CRISPR/Cas9的基因组编辑很重要。为了解决这个问题，我们构建并验证了三种基于荧光Cas9的报告基因，称为DSB-Spectrum，它们同时量化了DSB中多种DNA修复途径的贡献。DSB-Spectrum报告基因通过无错误典型非同源末端连接（c-NHEJ）与同源重组（HR;报告基因1）、诱变修复与HR（报告基因2）以及诱变末端连接与单链退火（SSA）与HR（报告基因3）区分DSB修复。使用这些报告，我们表明，抑制c-NHEJ因子DNA-PKcs增加修复的HR，但也大大增加诱变SSA。我们的数据表明，SSA介导的DSB修复也发生在内源性基因组位点，由Alu元件或同源基因区域驱动。最后，我们证明了长距离末端切除因子DNA 2和Exo 1促进SSA和降低HR，当两种途径竞争相同的底物时。这些新的基于Cas9的DSB-Spectrum报告基因有助于全面分析修复途径串扰和DSB修复结果。需要正确修复断裂的DNA分子以防止潜在的致癌突变。为了研究修复保真度和机制，货车de Kooij等人开发了单细胞报告基因，其检测DNA断裂是否通过无错误修复或诱变修复来固定。
DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome. Correct repair of broken DNA molecules is required to prevent potentially oncogenic mutations. To study repair fidelity and mechanism, van de Kooij et al. developed single cell reporters that detect if DNA breaks are fixed by error-free or mutagenic repair.
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影响因子：
14.9
作者：
Brinkman, Eva K.;Kousholt, Arne N.;van Steensel, Bas
通讯作者：
van Steensel, Bas
由 53BP1-RIF1 和 BRCA1-CtIP 组成的细胞周期依赖性调节回路控制 DNA 修复途径的选择。
影响因子：
16
作者：
Escribano-Diaz, Cristina;Orthwein, Alexandre;Durocher, Daniel
通讯作者：
Durocher, Daniel
影响因子：
21.3
作者：
Dev H;Chiang TW;Lescale C;de Krijger I;Martin AG;Pilger D;Coates J;Sczaniecka-Clift M;Wei W;Ostermaier M;Herzog M;Lam J;Shea A;Demir M;Wu Q;Yang F;Fu B;Lai Z;Balmus G;Belotserkovskaya R;Serra V;O'Connor MJ;Bruna A;Beli P;Pellegrini L;Caldas C;Deriano L;Jacobs JJL;Galanty Y;Jackson SP
通讯作者：
Jackson SP
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B
影响因子：
3.9
作者：
Flynn, Elizabeth K.;Kamat, Aparna;Chandrasekharappa, Settara C.
通讯作者：
Chandrasekharappa, Settara C.