Programmed DNA elimination in a unicellular model: coordination between DNA cleavage and double-strand break repair – CURE

DNA double-strand breaks (DSB), a threat for genome integrity, are repaired through two alternative pathways: homologous recombination or non-homologous end joining (NHEJ). Although potentially harmful, programmed DSB (prDSB) contribute to essential physiological processes (e.g. meiotic recombination, immunoglobulin gene assembly). We propose that physiological processes involving prDSB require tight coordination between DNA cleavage and DSB repair. With its two distinct nuclei, the ciliate Paramecium tetraurelia is a powerful unicellular model to test this hypothesis. The diploid micronucleus (MIC) hosts the germline genome that is transmitted to sexual progeny. The polyploid macronucleus (MAC), derived from the MIC at each generation, contains the rearranged somatic genome that is the substrate of gene transcription. During new MAC development, programmed genome rearrangements (PGR) eliminate ~30% of germline DNA, including DNA repeats and 45,000 Internal Eliminated Sequences (IESs). IES excision involves massive DSB introduction at IES boundaries, followed by precise NHEJ-mediated DSB repair.

The objectives of the CURE proposal focus on three unresolved challenges:

Objective 1. Biochemical and structural properties of the endonuclease complex.
Several components of the core endonuclease are known: PiggyMac (Pgm), the catalytic subunit, and five Pgm-like partners (PgmL1 to 5) that are required in vivo for DNA cleavage at IES ends. Whether they all form a large complex and how many subunits constitute the active endonuclease machinery have not been established. We will combine whole-cell immuno-staining with biochemical and biophysical methods to determine the stoichiometry, assembly order and organization of sub-complexes reconstituted from recombinant proteins or purified from Paramecium nuclei. We will set up in vitro functional assays and initiate structural analyses of Pgm-associated complexes. Finally, we will search for novel factors participating in endonuclease assembly.

Objective 2. Pgm association with its chromatin targets.
Eliminated sequences share no conserved motif, suggesting that Pgm does not bind its cleavage sites through recognition of a specific sequence. Its stable association with the nucleus requires protein partners (e.g. PgmLs). Non-coding RNAs also participate in targeting DNA for elimination. We will monitor Pgm association with chromatin using nuclear fractionation and whole-cell immunostaining, and correlate our data with genome-wide profiling of Pgm binding sites, after depletion of different factors (components of the endonuclease complex, chromatin and/or RNAs). To search for new chromatin-associating factors, we will analyze the protein and RNA content of chromatin fractions from purified new MACs.

Objective 3. Molecular and structural analyses of the coupling between DNA cleavage and DSB repair.
A specialized NHEJ factor (Ku70/Ku80c), containing one of three P. tetraurelia Ku80 homologs, is required to stabilize Pgm in the nucleus and activate IES end cleavage, indicative of strong coupling between DNA cleavage and DSB repair. To characterize the specificity of Ku80c, we will compare the structures of Ku70/Ku80a and Ku70/Ku80c and search for specific interactants of Ku70/Ku80c. We will test the effect of adding Ku to recombinant Pgm/PgmL complexes, at the structural and functional levels. We will also examine the role of other NHEJ factors in IES excision.

To achieve these objectives, two groups will bring together their highly complementary expertise. Partner 1 is a leader in Paramecium genomics, molecular and cellular biology, and biochemistry. Partner 2 is internationally renowned in the biophysical and structural analysis of DNA repair complexes. The CURE project will bring novel mechanistic insights into how Pgm, PgmLs and NHEJ factors interact with each other, recognize their substrates on chromatin, perform DNA cleavage and coordinate DSB repair during massive PGR.