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

• Applicant: Professor Dr. Holger Puchta
• Subject Area: Plant Genetics and Genomics
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 428712496

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.

• DFG Programme: Research Grants