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PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks

C Anne-Marie Couto1, Hong-Yu Wang, Joanna C A Green

  • 1Department of Biochemistry, University of Oxford, England, UK.

The Journal of Cell Biology
|August 3, 2011
PubMed

Insights

Poly adenosine diphosphate (ADP)-ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is crucial for DNA repair. This study reveals PARylation

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Poly adenosine diphosphate (ADP)-ribosylation (PARylation) is a key cellular response to DNA damage, particularly DNA double-strand breaks (DSBs).
  • Poly ADP-ribose polymerases (PARPs) catalyze PARylation, a post-translational modification essential for various DNA repair pathways.
  • Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major DNA repair mechanisms for DSBs, with their balance critical for genomic stability.

Purpose of the Study:

  • To investigate the role of PARylation in regulating nonhomologous end joining (NHEJ) in Dictyostelium discoideum.
  • To identify specific poly ADP-ribose polymerases (PARPs) involved in DNA damage response and their roles in NHEJ.
  • To elucidate the mechanism by which PARylation influences the recruitment and retention of DNA repair factors at DSBs.

Main Methods:

  • Utilizing Dictyostelium discoideum as a model organism to study DNA repair pathways.
  • Employing genetic disruption of PARP genes (Adprt1a, Adprt1b, Adprt2) to assess their roles in DNA damage resistance and repair.
  • Analyzing the impact of PARP disruption on NHEJ and homologous recombination (HR) efficiencies.
  • Investigating the recruitment and retention of Ku proteins at DSBs using biochemical and imaging techniques.
  • Mutational analysis of a PAR interaction motif in Ku70 to determine its functional significance.

Main Results:

  • PARylation occurs at single-strand breaks, requiring Adprt1b and Adprt2 for resistance.
  • Adprt1a and Adprt2, but not Adprt1b, are required for PARylation at DSBs.
  • Disruption of adprt2 had a minor effect on NHEJ, while adprt1a disruption impaired NHEJ and increased HR.
  • PARylation promotes NHEJ by enhancing the recruitment and/or retention of Ku at DSBs.
  • A PAR interaction motif in Ku70 is essential for PAR-dependent NHEJ regulation and efficient end joining.

Conclusions:

  • PARylation plays a novel, regulatory role in nonhomologous end joining (NHEJ) by influencing Ku recruitment to DNA double-strand breaks (DSBs).
  • Specific PARPs (Adprt1a, Adprt2) and a PAR-binding motif in Ku70 are critical for efficient PAR-dependent NHEJ.
  • These findings highlight the intricate interplay between PARylation and DNA repair pathways, offering insights into maintaining genomic integrity.

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