The role of DNA-protein crosslink repair in meiotic recombination, T-DNA integration and gene editing

虽然DNA-蛋白质交联(DPC)对基因组的完整性构成严重威胁，但直到最近，DPC修复的主要机制才在人类和酵母中被阐明。通过使用CRISPR/Cas9突变体，我们最近能够定义植物中DPC修复的途径。我们可以证明，通用修复蛋白酶SPRTN/Wss1(WSS1A)的同源基因对拟南芥的DPC修复是必不可少的。在植物中存在另外两条DPC修复途径，由酪氨酸二酯酶1(Tdp1)和DNA内切酶MUS81定义。有趣的是，在这些研究中，我们发现有迹象表明，DPC修复也是遗传过程所必需的，而它的作用以前从未被预期到：我们发现wss1A突变体有生育缺陷，并显示出被农杆菌转化的能力降低。我们推测，这些表型缺陷是由于可能是共价DNA蛋白中间体加工过程中的问题所致。根据目前的建议，一方面，我们希望通过分子和细胞学分析来确定WSSA1在胚胎发育中的分子作用。在wss 1a突变体中，雄性和雌性生殖系的可育性都降低了，我们能够在减数分裂重组过程中发现异常的中间产物。利用一组交叉的减数分裂突变体，我们现在想要阐明在减数分裂过程中WSS1A所需的特定步骤。我们还将分析杂交的数量是否发生了变化，这将通过花粉分型来测试。另一方面，通过测定T-DNA的转化效率和测序基因组T-DNA整合模式，我们想要阐明单个DPC途径在整合过程中从T-DNA中去除共价连接的VirD2和紧密结合的VirE2单链结合蛋白的详细作用。在这里，重要的是确定细胞的瞬时转化是否也受到阻碍，这将表明WSS1A在去除病毒E2中发挥了特定的作用。除病毒E2外，其他以非共价方式与DNA紧密结合的蛋白质也可能是WSS1A的底物，这表明DPC修复也可能影响基因编辑。由于细菌CRISPR/Cas核酸酶在DSB诱导过程中和之后与真核DNA紧密结合，它们的持续存在可能会影响修复途径的选择和修复模式。因此，我们还想通过应用我们成熟的基因编辑系统，使用来自化脓性链球菌和金黄色葡萄球菌的Cas9和来自沙门螺科细菌的Cas12a，来测试atwss1A突变体在非同源末端连接以及通过同源重组进行基因打靶的情况。因此，该项目的结果不仅有助于确定DPC修复在减数分裂重组和T-DNA整合中的作用，而且还可能导致基因编辑的改进。

Although DNA-protein crosslinks (DPCs) represent a severe threat to genome integrity, it was only recently that the main mechanisms of DPC repair were elucidated in humans and yeast. By the use of CRISPR/Cas9 mutants, we were lately able to define the pathways for DPC repair in plants. We could show that a homologue of the universal repair proteases SPRTN/WSS1 (WSS1A) is essential for DPC repair in Arabidopsis. Two further DPC repair pathways exist in plants, defined by the tyrosyl-DNA phosphodiesterase 1 (TDP1) and the DNA endonuclease MUS81. Interestingly, during these studies we found indications that DPC repair is also required for genetic processes where its role has not been anticipated before: We discovered that the wss1A mutant has a fertility defect and shows reduced transformability by Agrobacterium tumefaciens. We speculate that these phenotypic defects are due to problems in processing of presumably covalent DNA protein intermediates. With the current proposal, on one hand we want to define by molecular and cytological analysis the molecular role of WSSA1 in germ development. Fertility of both the male and female germline is reduced in the wss1a mutants and we were able to identify aberrant intermediates during meiotic recombination. Using a set of crossed in meiotic mutants, we now want to elucidate at which specific step during meiosis WSS1A is required. We will also anayze whether the number of crossovers is changed, which will be tested by pollen typing. On the other hand, by determining T-DNA transformation efficiencies and sequencing genomic T-DNA integration patterns, we want to elucidate the detailed roles of the individual DPC pathways in removing the covalently linked VirD2 and the tightly bound VirE2 single strand binding proteins from the T-DNA, during integration. It is important here to determine whether transient transformation of cells is also hampered, which would indicate a specific role for WSS1A in VirE2 removal. Other proteins tightly bound to DNA in a non-covalent manner, besides VirE2, could also be substrates for WSS1A, suggesting that DPC repair may also influence gene editing. As the bacterial CRISPR/Cas nucleases are tightly binding to eukaryotic DNA during and after DSB induction, their persistent presence might influence the choice of repair pathway, as well as repair patterns. Therefore, we also want to test the atwss1A mutant on non-homologous end joining, as well as on gene targeting by homologous recombination, by applying our well established gene editing systems using Cas9 from both S. pyogenes and S. aureus and Cas12a from Lachnospiraceae bacterium. Thus, the outcome of this project will not only help to define the role of DPC repair in meiotic recombination and T-DNA integration, but might also lead to improvements in gene editing.