Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
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DOI：
10.1101/2022.12.07.519501
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发表时间：
2022-12
影响因子：
16.6
通讯作者：
R. Deshpande;Alberto Marin-Gonzalez;T. Ha;T. Paull
中科院分区：
文献类型：
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作者：
R. Deshpande;Alberto Marin-Gonzalez;T. Ha;T. Paull
Mre 11-Rad 50-Nbs 1（MRN）复合物通过短距离去除5条双链来识别和处理DNA双链断裂，以进行同源重组。由MRN进行的内切核酸溶解加工需要在断裂位点稳定结合的蛋白质-我们假设哺乳动物中的DNA依赖性蛋白激酶（DNA-PK）发挥作用。在这里，我们通过分离和测序DNA-PK结合的DNA片段（MRN切割的产物）来询问MRN依赖性加工的位点。当DNA-PK被催化阻断时，这些中间体以最高的效率产生，在断裂位点的200 bp内产生产物，而在不存在激酶抑制的情况下，DNA-PK产物显示出更大的分散性。使用光激活的Cas9诱导断裂有助于DNA-PK和Mre 11结合的时间分辨，表明Mre 11和DNA-PK都在释放DNA-PK结合产物之前与DNA末端结合。这些结果支持一个连续的模型，涉及同源和非同源修复复合物之间的协同作用的双链断裂修复。
The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.