Novarina Daniele, Desai Ridhdhi, Vaisica Jessica A, Ou Jiongwen, Bellaoui Mohammed, Brown Grant W, Chang Michael
European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, the Netherlands.
Department of Biochemistry and Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.
G3 (Bethesda). 2020 Jun 1;10(6):1853-1867. doi: 10.1534/g3.120.401137.
Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionarily conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.
同源重组是维持基因组完整性的重要机制，多种癌症及癌症易感综合征中存在多个同源重组基因发生突变的情况。然而，由于在某些情况下同源重组会导致诱变结果，因此该途径必须受到严格调控，而有丝分裂超重组是基因组不稳定的一个标志。我们针对缺失时会导致直接重复序列之间超重组的基因进行了两项筛选。一项是采用经典的菌斑和影印培养法进行的。另一项是采用一种旨在检测低频事件的高通量复制固定技术进行的。这种方法使我们能够独立于其开发时所处的复制性衰老背景来验证高通量复制固定方法。此外，通过结合这两种方法，我们能够鉴定并验证35个缺失会导致自发直接重复序列重组增加的基因。其中包括错配修复基因、Sgs1-Top3-Rmi1复合物、核糖核酸酶H2复合物、参与氧化应激反应的基因以及许多其他DNA复制、修复和重组基因。由于我们筛选出的多个基因在进化上是保守的，并且重复元件在哺乳动物基因组中占很大比例，因此我们的工作可能与理解人类基因组完整性的维持有关。
