Hartlerode Andrea J, Willis Nicholas A, Rajendran Anbazhagan, Manis John P, Scully Ralph
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America.
Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS Genet. 2016 Nov 10;12(11):e1006410. doi: 10.1371/journal.pgen.1006410. eCollection 2016 Nov.
A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer.
哺乳动物细胞中一部分同源重组（HR）事件通过“长片段”基因转换得以解决，这反映出在终止之前从供体姐妹染色单体复制了几千个碱基对。缺乏主要遗传性乳腺癌/卵巢癌易感基因BRCA1或BRCA2的细胞，或某些其他HR缺陷型细胞，表现出有利于长片段基因转换的偏向性，这表明这种异常的HR结果可能与基因组不稳定有关。如果基因转换的终止发生在与断裂的第二个末端缺乏同源性的区域，那么通过退火（即同源配对）进行HR终止的正常机制就不可用了，终止必须通过尚未明确的非经典机制发生。在这里，我们使用先前描述的HR报告基因来分析哺乳动物细胞中长片段基因转换的非经典终止机制。我们发现，在没有经典的非同源末端连接基因XRCC4的情况下，非经典HR终止也可以发生。我们观察到在与断裂的第二个末端重新连接的过程中，必须使用微同源性（MH）介导的末端连接和/或核苷酸添加。值得注意的是，非经典HR终止与复杂的断点有关。我们确定了同源性介导的模板切换以及潜在的MH介导的模板切换/微同源性介导的断裂诱导复制在非经典HR终止位点复杂断点形成中的作用。这项工作确定非经典HR终止是基因组不稳定和癌症中复杂断点形成的一个潜在因素。
