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Nucleases in homologous recombination: from basic principles to genome editing

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

DNA of all living organisms is prone to damage. DNA breaks represent one of the most toxic forms of DNA damage. Cell can employ either of two main DNA double-strand break repair pathways, non-homologous end-joining or homologous recombination. In end joining, the two broken pieces of DNA are joined without a template, which may lead to point mutations or joining of wrong DNA fragments, leading to translocations. The other process, termed homologous recombination is more accurate, but also a more complex system that necessitates multifaceted regulatory mechanisms.

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.
Aim I: We have been using both yeast and human systems to understand the mechanisms of DNA break processing. Our initial results identified Sae2 as a regulator of the nuclease activity of the MRX complex (in yeast) and correspondingly CtIP as a regulator of the human MRN (in human cells). The initial processing of DNA breaks by the Mre11/MRE11 nuclease (within the Mre11-Rad50-Xrs2 - MRX - or MRE11-RAD50-NBS1 - MRN - complex) is particularly important for the processing of DNA ends with noncanonical structures (i.e. hairpins, covalently attached proteins). In subsequent work, we defined how phosphorylation of Sae2 or CtIP by CDK activates the endonuclease of MRX/MRN, which initiates DNA end resection early in the cell cycle (S phase). We have also uncovered an interplay of CtIP with DNA2 acting in the long-range resection acting downstream of MRN. We were able to create separation of function mutants of CtIP that activates either MRE11 or DNA2 nuclease, and analyzed the phenotypes of the mutants in cellular assays.

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.
Our work on DNA end resection not only clarified how resection functions on the mechanistic level, but uncovered how resection is regulated. We helped explain how phosphorylation of CtIP/Sae2 activates short-range resection early in cell cycle, and how phosphorylation of CtIP by PLK late in the cell cycle inhibits downstream long-range resection. One of the additional unexpected results obtained so far relates to the relationship of resection and Cas9. Our data indicating that Cas9 needs to be actively displaced from DNA breaks to initiate their repair is broadly relevant for the gene editing field, well beyond the interests related to homologous recombination.

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.
Summary Aim II