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

在所有生物的基因组中，单链断裂(SSB)是一种比双链断裂(DSB)更常见的DNA损伤。由于高度特异的内切酶的存在，在过去的25年里，DSB修复在多细胞真核生物中得到了非常详细的分析。这不仅适用于修复机制，也适用于所涉及的蛋白质。直到最近，通过使用CRISPR/Cas系统才有可能在任何基因组位置高效地诱导独特的SSB。利用这项技术，目前的建议旨在阐明模式植物拟南芥SSB修复的性质。我们已经能够证明，单个SSB可以极大地诱导重复序列之间的同源重组(HR)。在目前的项目中，我们将通过应用现有的突变体来分析哪些修复和重组蛋白参与了SSB诱导的植物修复途径。正如我们之前已经证明的那样，T DNA可以整合到预先形成的DSB中，我们想要测试SSB是否也可以特异性地启动T DNA整合位点。此外，与唯一的SSB相比，我们发现在两个相反的DNA立场上距离较近(高达100个核苷酸)的两个SSB具有高度的诱变性，产生缺失并通过填充合成插入来实现。我们现在的目标是确定导致不同突变类别形成的植物因素。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诱导突变DNA修复的偶联SSB。为此，我们将在从100bps到2kb(如果需要，甚至更长)的不同距离上引发SSB，从而导致3或5个悬垂。我们将测试欧盟和异色区。除了阐明植物SSB修复的基本机制外，本研究结果还将对基于Cas9尼克酶的植物基因组工程的发展具有重要的指导意义。

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.