Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication
Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication
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DOI：
10.1371/journal.pgen.1008828
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发表时间：
2020-07-01
期刊：
影响因子：
4.5
通讯作者：
McCulloch, Richard
中科院分区：
文献类型：
--
作者：
Damasceno, Jeziel D.;Reis-Cunha, Joao;McCulloch, Richard
同源重组在细胞分裂过程中的基因组维持中起着关键作用，但指导反应的因子的丧失在任何微生物中都没有被描述为致命的。在这里，我们使用了一种遗传策略来选择性地诱导利什曼病中5个在同源重组中起作用的基因的单一和双重丢失，揭示了两件事。首先，与催化同源重组的RAD 51相关的任何基因的丢失不是立即致命的，而是导致增加的生长障碍和基因组损伤累积。第二，RAD 51的缺失导致利什曼原虫基因组复制程序的显著改变。因此，我们发现利什曼原虫的同源重组是必不可少的，部分原因是它在基因组传递中发挥了意想不到的作用。同源重组（HR）与基因组复制有着密切的关系，无论是在可能阻止DNA合成的DNA损伤修复过程中，还是在处理复制叉的停顿时。最近的研究使我们想知道HR是否可能在复制真核寄生虫利什曼原虫的基因组中发挥更重要的作用。关于HR基因是否必不可少的证据已经出现，全基因组作图为DNA复制起始位点（称为起源）的非正统组织提供了证据。为了回答这个问题，我们采用了CRISPR/Cas9和DiCre相结合的方法来快速生成和评估条件性消融RAD 51和三种RAD 51相关蛋白在利什曼原虫中的作用。使用这种方法，我们证明了这些HR因子中的任何一种的损失都不会立即致命，但在每种情况下，生长都会随着时间的推移而减慢，并导致DNA损伤和具有异常DNA含量的细胞的积累。尽管有这些相似之处，我们发现只有RAD 51或RAD 51 -3的缺失会损害DNA合成，并导致全基因组突变水平升高。此外，我们表明，这两个HR因子以不同的方式起作用，因为RAD 51的消融，而不是RAD 51 -3，对DNA复制有深远的影响，导致在主要起点的起始损失和亚端粒的DNA合成增加。我们的工作澄清了有关HR对利什曼原虫生存的重要性的问题，并揭示了RAD 51在微生物真核生物基因组复制程序中的意想不到的核心作用。
Author summary Homologous recombination plays a key role in genome maintenance during cell division, but loss of factors directing the reaction has not been described as being lethal in any microbe. Here, we have used a genetic strategy to selectively induce loss, singly and doubly, of five genes inLeishmaniathat act in homologous recombination, revealing two things. First, loss of any gene related to RAD51, which catalyses homologous recombination, is not immediately lethal, but leads to increasing growth impairment and genome damage accumulation. Second, loss of RAD51 causes a pronounced change in the programme ofLeishmaniagenome replication. Thus, we show that homologous recombination inLeishmaniacan be essential, in part due to an unanticipated role in genome transmission.Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome ofLeishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins inLeishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival ofLeishmaniaand reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.