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Nucleases in homologous recombination: from basic principles to genome editing

Periodic Reporting for period 3 - HRMECH (Nucleases in homologous recombination: from basic principles to genome editing)

Reporting period: 2019-10-01 to 2021-03-31

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 is 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 is 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 to define the function of a key meiotic nuclease complex that functions in this step. This will help explain how cells manage to undergo meiotic division, generate diversity and avoid aneuploidy.
Aim I: We have been using both yeast and human recombinant proteins to understand the mechanism of DNA end resection. Upon DNA breakage, DNA end resection is a necessary step that makes the broken DNA end compatible with homology-directed repair, and involves several nuclease complexes.

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 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 regulates its capacity to promote the MRX/MRN nuclease complexes. We were able to show that in yeast, Sae2 phosphorylation regulates its oligomeric state and interaction with Rad50, which affects the nuclease activity of the ensemble. In human cells instead, the phosphorylation of CtIP mostly regulates MRE11 via its interplay with NBS1. We also defined how DNA end binding proteins (most notably Ku) functionally interact with the MRX-Sae2 ensemble, and how they direct DNA degradation to the 5'-terminated DNA strand. We are now working on understanding how CtIP regulates other nuclease complexes downstream of MRE11. The work from these experiments was published in journals including Molecular Cell, EMBO J, Genes and Development, PNAS and Nature Communications, and we have two manuscripts under review (PNAS and NAR).

Aim II: We have been working with human recombinant proteins to understand how the MLH1-MLH3 nuclease processes recombination intermediates. In meiosis, the MLH3 nuclease is required for proper chrososome seggregation and exchange of DNA segments between maternal and paternal chromosomes. How is the MLH3 nuclease activated is not clear.

We defined the MLH1-MLH3 nuclease activity in vitro and found that it is stimulated by MSH4-MSH5, EXO1 and RFC-PCNA. We believe that assymetric presence of theseco-factors of MLH1-MLH3 may direct biased processing of recombination intermediates into crossover recombination products. We are currently preparing a manuscript for submission. Beyond this work, we have been involved in a number of collaborative projects, which have been published (Genes and Development, eLife) or are in preparation.
In the remaining period of the ERC funding, we aim to further study DNA end resection (Aim I), namely the interplay of the short-range resection complexes (MRX-Sae2 or MRN-CtIP) with nucleases that function downstream, including EXO1 or DNA2, in both yeast and human systems. We observed that CtIP promotes the DNA2 nuclease; the first manuscript is currently under review in PNAS. We identitified sevelar DNA2 mutants that do not interact with CtIP and intend to analyze the associated cellular phenotypes. We are also investigating the function of the BRCA1-BARD1 proteins in DNA end resection.

Next, we aim to define whether resection and loading of RAD51, the main strand exchange protein, is coordinated. This is the case in E. coli, where the RecBCD complex both resects DNA and loads RecA (RAD51 ortholog). In eukaryotic systems, such coordination has not been described to-date, although RAD51 physically interacts with several components of the resection complexes. We will investigate whether resection complexes facilitate RAD51 loading. This might allow us to couple DNA end resection with strand exchange reactions in vitro, and learn thus about the underlying mechanisms.

Regarding Aim II, our work with MLH1-MLH3 opened doors to further investigate this specialized nuclease complex. This will include work to define whether specific secondary DNA structures, strand discontinuities, posttranslational modifications or additional protein co-factors affect DNA cleavage. Beyond meiotic recombination, the MLH1-MLH3 complex has a residual mismatch repair activity and affects the stability of DNA repeats. This is likely relevant for a subset of colon cancers and several neurogenerative diseases. We aim to define the function of MLH1-MLH3 in mismatch repair as well.
Cartoon summarizing the results obtained so far within the HRMECH project