4 Year MRC PhD Programme: New mechanisms of branched DNA molecule processing during cytokinesis: Working out the role of the conserved LEM-3 nuclease; from C. elegans to mammals

SUMMARY AND WORK HYPOTHESIS The maintenance of genome stability is essential for cellular and organismal survival, and a failure in genome maintenance results in mutagenesis, leading to cancer and inherited disease. ‘Branched’ DNA molecules that arise, when replication is incomplete and/or as part of DNA repair processes, can lead to the formation of chromatin bridges between dividing cells. The last-minute processing of branched DNA that persists until cytokinesis and results in chromatin bridges remains largely unexplored.

Our working hypothesis is that the LEM-3 nuclease is dynamically localized to the midbody to processes branched DNA structures just before the completion of cytokinesis.

AIMS AND EXPERIMENTAL PLAN:

How does the LEM-3 nuclease resolve chromatin bridges during cytokinesis to maintain genome integrity?
We want to further 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. We will use high-resolution real time imaging of chromatin. We want to determine if and how the conserved Aurora A kinase regulates LEM-3 localization and activity.

B. What kind of branched DNA structures are cleaved by LEM-3, and how does cleavage occur?

LEM-3 has in vitro nuclease activity, but its specificity is not known. We want to uncover the DNA structures, which are cleaved by LEM-3, to determine LEM-3 reaction mechanisms and to work towards determining the structure of LEM-3 (the student will interact with the Lilley lab).



What is the role of the Ankle-1 in vertebrates.
Lem3/Ankle1 is highly conserved and given the enormous fidelity of genome maintenance, DNA repair pathways are often redundant, especially in mammals. We will determine if Lem3 is also located at the midbody of mammalian cells, and if so if this is regulated as in C elegans. We will generate antibodies against human LEM3/Ankle-1 and using Cas-9 gene editing will also generate appropriate GFP fusions.

The students will undertake an interdisciplinary project raging from C. elegans and human tissue culture in vitro and in vivo procedures (Gartner and Rouse lab) to advanced biochemistry and enzymatic (nuclease) reaction mechanisms.