Processing of branched DNA molecules during cytokinesis, uncovering new mechanisms of genome maintenance

In each of the ~1014 cells of the human body, the genetic material (DNA) is composed of two times 3 billion 'nucleotide-letters' corresponding to a length of 3 meters. Remarkably we synthesize the equivalent of a light year of DNA during our live time. The genetic code has to be kept intact. However, DNA molecules are highly reactive in the intracellular milieu. Typically, in each cell of our body, thousands of DNA lesions occur daily, all of which could lead to mutations. Before cells ultimately divide, all DNA lesions have to be repaired in order to ensure the accurate maintenance of the genome. Thus, to ensure faithful genome maintenance mechanisms multiple fail safe mechanisms exist. Failure of genome maintenance leads to mutation, and this can result in cancer, premature ageing, neurodegeneration and inherited disease.

To ensure correct genome maintenance, chromosomes have to be properly segregated to daughter cells. This requires the removal of all connections between separating chromosomes (sister chromatids) before cells ultimately divide. DNA bridges between segregating chromosomes can arise when DNA replication is not complete, when unresolved 'joint' or 'branched' DNA repair intermediates persist, or when chromosomes fail to properly condense. Failure to process chromatin bridges when cells divide leads to genome instability, aneuploidy and polyploidization. Aneuploidy refers to the loss or gain of entire chromosomes; polyploidization is the term describing the inappropriate duplication of the entire genome. Both aneuploidy and polyploidization commonly occur in cancer cells. It was known for more than a decade that DNA bridges that link dividing cells can delay the completion of the final step of cell division referred to as cytokinesis. This cytokinesis delay was termed 'NoCut checkpoint' more than 10 years ago. However, it was not known how DNA bridges are processed just before cytokinesis to allow for faithful chromosome segregation and genome maintenance.

We found that the LEM-3 (Ankle1 in vertebrates ) nuclease defines a new genome integrity mechanism by processing DNA bridges immediately before cells divide. LEM-3 acts at the midbody, the structure where cells finally divide at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 processes chromatin bridges that arise from a variety of perturbation on DNA metabolism. These include incomplete DNA replication, incomplete DNA repair or the perturbation of chromosome structure. Thus LEM-3 affords cells with a last chance saloon mechanism to process multiple types of DNA bridges just before cells divide. Strikingly, LEM-3 synergizes with BRC-1/BRCA1, a protein commonly mutated in patients suffering from breast and ovarian cancer and involved in mending DNA damaged by homologous recombination, to promote genome integrity after DNA damage. These findings provide a molecular basis for the suspected role of human LEM-3 look-like Ankle1 in human breast cancer.

Here we propose to elucidate the molecular mechanisms that underlie LEM-3 function. Our studies take advantage of the small nematode C. elegans as a model. This is the most cost- and time-efficient approach and DNA response mechanisms are conserved in evolution. At the same time we shall extend our studies to the role of Ankle1 in genome stability in mammalian cells.

We will ask the following questions:

I) What types of DNA structures are processed by the LEM-3 nuclease and what are the reaction mechanisms?

II) What are the mechanisms that control LEM-3 association with the midbody during cytokinesis?

III) What other factors regulate LEM-3 to resolve branched DNA intermediates during cytokinesis?

IV) What is the role of Ankle1 in vertebrates and how does it function?

We want to investigate how DNA bridges induced by under-replication, by recombination intermediates and by chromosome condensation defects are processed by LEM-3 (Ankle1 in vertebrate cells) just before the completion of cytokinesis.

Our objectives are as follows. We want to address:

I) What types of DNA structures are processed by the LEM-3 nuclease and what are the reaction mechanisms? We found that LEM-3 is involved in processing chromatin bridges induced by a variety of conditions, including incomplete DNA replication, unprocessed DNA recombination intermediates and incomplete chromosome condensation. We want to understand which branched DNA intermediates are cleaved by LEM-3 and how this works.


II) What are the mechanisms that control LEM-3 association with the midbody during cytokinesis? We want to understand how LEM-3 functions as part of a fundamentally new mechanism of genome maintenance by processing chromatin bridges at the midbody just before cells divide and how this is regulated by the conserved Aurora B/AIR-2 kinase.


III) What other factors regulate LEM-3 to resolve branched DNA intermediates during cytokinesis? We want to search for factors required for appropriate LEM-3 activation, using both genetic and biochemical approaches


IV) What is the role of Ankle1 in vertebrates and how does it function? We aim to find DNA repair defects of ankle1 mutants, in conjunction with the inactivation of the brca1, slx1 and mus81 repair genes, and we will test if Ankle1 acts like LEM-3 by processing chromatin bridges during cytokinesis.

The human genome is comprised of three billion nucleotides, which comprise our genetic information. This information has to be kept intact. The maintenance of genome integrity is essential for cellular and organismal survival and genetic disease, ageing, many neurodegenerative conditions and cancer are all correlated with increased levels of genome instability.

Academic Impact

Here we investigate, a fundamentally new mechanism for DNA repair, by studying how a structure specific nuclease termed LEM-3 in C. elegans and Ankle 1 in human cells allows for mending a variety of DNA structures associated with multiple perturbation of DNA metabolism just before cells divide. Our studies are important as mutations, which are passed on to daughter cells are fixed and have the potential to lead to the cancerous transformation of affected cells. Furthermore, when DNA intermediates are not resolved before cells divide chromosome fusions and the duplication of the entire genome within a single cell may occur.

Mid and long term Societal and Commercial Impact

Cancer a disease responsible for more than 1/4 of all deaths in the UK is caused by genome instability. Hence, exploiting the inherent genome instability of cancer cells to target their selective elimination provides a new inroad into cancer therapy. Intriguingly, a small area of the human genome including the LEM-3 orthologue Ankle1 was associated with increased susceptibility to breast and ovarian cancer carrying Brca1 and Brca2 mutations. Given that we demonstrated that lem-3; brca-1 double mutants show increased sensitivity to DNA damage this is consistent with Ankle1 being the most likely gene to affect breast cancer disposition. Cancer cells are commonly treated with genotoxic agents to affect their killing by inflicting excessive DNA damage, but such treatment, which includes radiotherapy, equally affects healthy cells, reducing the therapeutic window and causing excessive side effects. DNA lesions are eliminated by multiple redundant (fail save) DNA repair modalities. This redundancy can be exploited for cancer treatment if cancer cells are defective in one of these redundant pathways. For instance mutation of Brca1 and Brca2 and other DNA repair mutations accounts for ~20% of all breast cancer case. Thus targeting the Ankle1 nucleases by specific drugs has a tremendous potential. We aim to do exactly this in the mid- (5 years) and long term (10-15 years). Our research will provide the basis for setting up high throughput screens for compounds that selectively inhibit the Ankl1 nuclease. We will collaborate with the Dundee Drug Discovery Unit to do this work. Dundee has multiple interactions with the pharmaceutical industry and through collaboration and out-licencing we will facilitate drug discovery that will have an impact for cancer patients.


Teaching and Public Engagement

We are committed to train the next generation of researchers. Many of our former PhD students and postdocs run independent research groups. As such we contribute to UK higher education and economy. We act in the strong belief that competitive academic research cannot and should not be separated from teaching and mentoring. We thus take part in undergraduate and graduate teaching and we are involved in multiple outreach activities. Importantly, the Principal Investigator also organizes a Summer School to allow >30 students a hands-on experience in internationally competitive research laboratories.

Securing International Funding.

This project is part of our long-term collaboration between the Gartner, Rouse and Lilley laboratories focused on structure specific nucleases. This project will strengthen our case for a (~£10 million) EU Synergy application in late 2018 or 2019, in conjunction with the Dundee Drug Discovery Unit, aimed to systematically establish drug discovery pipelines for multiple nucleases including Ankl1, Slx1, Mus81 and Gen1.