Elucidating the mechanisms of DNA single strand break (SSB) repair in plants

在所有生物体的基因组中，单链断裂（SSBs）是一种比双链断裂（DSBs）更常见的DNA损伤。由于高特异性内切酶的可用性，在过去的25年中，人们对多细胞真核生物的DSB修复进行了非常详细的分析。这既适用于修复机制，也适用于相关的蛋白质。直到最近，使用CRISPR/Cas系统才有可能在任何基因组位点诱导高效独特的ssb。利用这一技术，目前的建议旨在阐明模式植物拟南芥中SSB修复的本质。我们已经能够证明单个SSBs在重复序列之间急剧诱导同源重组（HR）。在目前的项目中，我们现在将通过应用现有的突变体来分析修复和重组蛋白在植物中参与SSB诱导的修复途径。正如我们之前所展示的那样，T DNA可以整合到预形成的dsb中，我们想测试ssb是否也可以特异性地启动T DNA整合位点。此外，与一个独特的SSB相比，我们发现，在两个相反的DNA展台上，两个距离更近（高达100 nts）的SSB具有高度的诱变性，也会产生缺失和通过填充合成插入。我们现在的目标是确定导致不同突变类形成的植物因素。另一个重要的问题是，两个相邻的ssb必须相距多远才能不再被细胞识别为偶联的ssb，从而诱导诱变DNA修复。为此，我们将在从100 bps到2 kb（如果需要甚至更长）的不同距离上诱导ssb，从而导致3或5个悬垂。我们将测试eu和异色区域。本研究结果除了阐明植物中SSB修复的基本机制外，还将对基于Cas9缺失酶的植物基因组工程的发展具有重要意义。

Single strand breaks (SSBs) are a much more common DNA lesions than double strand breaks (DSBs) in the genomes of all organism. Due to the availability of highly specific endonucleases, DSB repair has been analyzed in great detail over the last 25 years in multicellular eukaryotes. This applies for the repair mechanisms as well as for the proteins involved. Only recently it became possible by the use of the CRISPR/Cas system to induce highly efficiently unique SSBs at any genomic site. Using this technology the current proposals aims to elucidate the nature of SSB repair in the model plant Arabidopsis thaliana. We were already able to show that that a single SSBs drastically induces homologous recombination (HR) between repeated sequences. In the current project we will now analyze by application of available mutants which repair and recombination proteins are involved in this SSB induced repair pathway in plants. As we could show previously that T DNA can integrate into preformed DSBs, we want to test whether also SSBs can initiate T DNA integration site specifically. Moreover, in contrast to a unique SSB, we found out that two SSBs in closer proximity (up to 100 nts) to each other in the two opposite DNA stands are highly mutagenic, producing deletions and by fill in synthesis insertions, too. We are now aiming to define the plant factors responsible for the formation of the different mutant classes. Another important question is how far two adjacent SSBs have to be apart that they are no longer recognized by the cell as coupled SSBs inducing mutagenic DNA repair. For that we will induce SSBs at various distances from 100 bps to a 2 kb (or even longer if required) that result in either 3 or 5 overhangs. We will test eu and heterochromatic regions. Besides elucidating the basic mechanism of SSB repair in plants the results of this study will also be highly relevant for the development of Cas9 nickase based genome engineering in plants.