ABSTRACT

Severe cellular sensitivity and aberrant chromosomal rearrangements in response to DNA interstrand crosslink (ICL) inducing agents are hallmarks of Fanconi anemia (FA) deficient cells. These phenotypes have previously been ascribed to inappropriate activity of non-homologous end joining (NHEJ) rather than a direct consequence of DNA ICL repair defects. Here we used chemical inhibitors, RNAi, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 to inactivate various components of NHEJ in cells from FA patients. We show that suppression of DNA-PKcs, DNA Ligase IV, and 53BP1 is not capable of rescuing ICL-induced proliferation defects and only 53BP1 knockout partially suppresses the chromosomal abnormalities of FA patient cells.

摘要

严重的细胞敏感性和响应 DNA 链间交联 (ICL) 诱导剂的异常染色体重排是范可尼贫血 (FA) 缺陷细胞的标志。这些表型先前已被归因于非同源末端连接 (NHEJ) 的不当活动，而不是 DNA ICL 修复缺陷的直接结果。在这里，我们使用化学抑制剂、RNAi 和成簇规则间隔短回文重复序列 (CRISPR)-Cas9 来灭活 FA 患者细胞中 NHEJ 的各种成分。我们表明抑制 DNA-PKcs、DNA 连接酶 IV 和 53BP1 不能挽救 ICL 诱导的增殖缺陷，只有53BP1敲除部分抑制了 FA 患者细胞的染色体异常。