Periodic Reporting for period 4 - HRMECH (Nucleases in homologous recombination: from basic principles to genome editing)
Reporting period: 2021-04-01 to 2022-03-31
The overall aim of the project was to understand the mechanism of function of nucleases that function in the homologous recombination pathway. Nucleases act both early and late during recombination. The early function involves a process termed DNA end resection. Here, nucleolytic processing of the DNA ends commits the repair to the recombination pathway, as resected DNA end are no longer ligatable by end-joining. The first aim of the project was to understand how nucleases function in DNA end resection, and how this contributes to the pathway choice in DNA double-strand break repair.
Understanding the regulation of the pathway choice is very relevant with regard to gene editing, as only a recombination-based mechanism can introduce a specific mutation. Understanding processes that affect the balance between the two main DNA double-strand break repair pathways can thus improve the efficacy of genome editing. The resection nuclease complexes have also a poorly-defined function at challenged DNA replication forks, and were linked to chemoresistance of certain types of cancer.
The second aim of the project is to define the function of a specific nuclease complex that functions late in recombination to separate recombining DNA molecules. In meiosis, homologous recombination has a specialized function to exchange genetic information between maternal and paternal DNA molecules, which helps proper chromosome segregation and promotes genetic diversity. Our aim was to define the function of a key meiotic nuclease complex that functions in this step. We helped explain how cells manage to undergo meiotic division, generate diversity and avoid aneuploidy.
We also studied the interplay of DNA end resection with Cas9 nuclease. We observed that Cas9 breaks are initially invisible to the resection machinery, because Cas9 bridges broken DNA. Cas9 thus needs to be actively displaced to initiate break repair. We are in the process of identifying ATP-driven DNA translocases that help dislocate Cas9 from break sites to initiate resection and repair. The work from these experiments (Aim I) was published in journals including Molecular Cell, Genes and Development, PNAS and Nature Communications.
Aim II: We studied the activation of the MLH1-MLH3 endonuclease, which is thought to process meiotic recombination intermediates. We could show that human MLH1-MLH3 is indeed a nuclease and that it is regulated by MSH4-MSH5, EXO1 and RFC-PCNA, resembling thus somewhat mismatch repair reactions. The main part of the project was published in Nature. Beyond this work, we have been involved in a number of collaborative projects, which have been also published (Genes and Development, eLife, PNAS) or are in preparation.
Regarding Aim II, our work with MLH1-MLH3 opened doors to further investigate this specialized nuclease complex. We will be able to use specific DNA substrates with secondary DNA structures, and precisely monitor DNA cleavage positions. It is expected these experiments will help us refine models explaining crossover-biased processing of recombination intermediates.