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Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome

Emeline Dubois1, Nathalie Mathy1, Vinciane Régnier1,2

  • 1Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.

Nucleic Acids Research
|January 21, 2017
PubMed

Insights

PiggyMac (Pgm) mediates DNA elimination in Paramecium. The catalytic DDD triad and cysteine-rich domains are essential, while the coiled-coil domain is crucial for multiple Pgm subunits to function in DNA excision.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • During sexual reproduction, the ciliate Paramecium undergoes programmed DNA elimination, removing 25-30% of its germline DNA.
  • This process involves the precise excision of approximately 45,000 internal eliminated sequences (IESs) from the somatic genome.

Purpose of the Study:

  • To investigate the functional roles of different domains within the PiggyMac (Pgm) protein, a domesticated transposase essential for IES excision.
  • To elucidate the molecular mechanisms underlying DNA cleavage and excision mediated by Pgm during Paramecium sexual processes.

Main Methods:

  • An in vivo complementation assay was developed using PiggyMac-Green Fluorescent Protein (Pgm-GFP) fusions.
  • Mutant Pgm variants lacking specific domains (catalytic DDD triad, cysteine-rich, coiled-coil) were expressed in Paramecium cells depleted of endogenous Pgm.

Main Results:

  • The catalytic DDD triad and the cysteine-rich (CR) domain are indispensable for Pgm activity, with mutations exhibiting a dominant-negative effect.
  • A mutant lacking the C-terminal coiled-coil (CC) domain showed partial activity with limiting Pgm but was inactive when Pgm was absent, suggesting a role in complex formation.

Conclusions:

  • IES excision in Paramecium is a multi-subunit process requiring the cooperative action of Pgm proteins.
  • At least a fraction of the active Pgm complexes must contain the CC domain for efficient DNA excision during sexual reproduction.

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