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

单链断裂是生物体基因组中比双链断裂更常见的DNA损伤。由于高度特异性核酸内切酶的可用性，在过去的25年中，在多细胞真核生物中已经非常详细地分析了DSB修复。这适用于修复机制以及所涉及的蛋白质。直到最近，通过使用CRISPR/Cas系统才有可能在任何基因组位点高效诱导独特的SSB。利用这项技术，目前的建议，旨在阐明SSB修复的模式植物拟南芥的性质。我们已经能够证明，一个单一的SSB会显著诱导重复序列之间的同源重组（HR）。在当前的项目中，我们现在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植物中SSB诱导的修复途径。正如我们之前所展示的，T DNA可以整合到预先形成的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，与独特的SSB相反，我们发现在两个相对的DNA链中彼此更接近（高达100 nt）的两个SSB是高度致突变的，产生缺失并通过合成插入来填充。我们现在的目标是确定负责形成不同的突变类型的植物因子。另一个重要的问题是两个相邻的SSB必须分开多远，它们不再被细胞识别为诱导诱变DNA修复的偶联SSB。为此，我们将在从100 bps到2 kb（或甚至更长，如果需要的话）的各种距离处诱导SSB，这导致3或5个突出端。我们将测试eu和异色区域。除了阐明植物中SSB修复的基本机制外，本研究的结果还将与植物中基于Cas9切口酶的基因组工程的开发高度相关。

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.