Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Precise excision of internal eliminated sequences (IESs) from the somatic genome is critical in ciliates like Paramecium.
• DNA double-strand breaks (DSBs) are introduced by PiggyMac endonuclease and repaired via non-homologous end joining (NHEJ).
• Ku70/80 proteins are essential for NHEJ, as their absence inhibits DNA cleavage.

Purpose of the Study:

• To investigate the role of Ku70/80 proteins in DNA double-strand break (DSB) repair during IES excision.
• To uncouple DNA cleavage and repair steps in Paramecium genome processing.
• To identify the consequences of impaired DSB repair on IES excision and genome integrity.

Main Methods:

• Utilized a DNA-binding-deficient Ku70 mutant (Ku70-6E) to separate DNA cleavage from repair.
• Co-expressed wild-type and mutant Ku70 to modulate NHEJ pathway activity.
• Employed high-throughput sequencing to analyze the developing macronuclear genome.

Main Results:

• Expression of Ku70-6E permitted DNA cleavage but resulted in unrepaired DSBs.
• Genome sequencing revealed de novo telomere additions at healed extremities.
• Numerous translocations occurred between sequences flanking the excised IESs.
• Coupling cleavage and repair ensures proper processing, highlighting the role of DSB repair proteins.

Conclusions:

• Ku70/80 proteins are essential for the concerted repair of DSBs during IES excision in Paramecium.
• The NHEJ pathway plays a crucial role in maintaining genome integrity post-cleavage.
• DSB repair proteins are implicated in the assembly of a synaptic precleavage complex, linking repair to initial cleavage events.