DNA-dependent conformational changes in the Ku heterodimer.

Area of Science:

• Molecular biology
• Biochemistry
• DNA repair mechanisms

Background:

• Ionizing radiation causes DNA double-strand breaks, necessitating repair via the nonhomologous end joining (NHEJ) pathway.
• The NHEJ pathway is initiated by Ku binding to DNA ends, facilitating interaction with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form an active kinase complex.
• The precise molecular mechanisms governing DNA-PK activation remain largely unknown.

Purpose of the Study:

• To investigate the influence of DNA binding on the structural conformation of the Ku heterodimer.
• To elucidate the role of structural changes in Ku in the activation of the DNA-PK complex.

Main Methods:

• Chemical modification using NHS-biotin to identify reactive sites.
• Mass spectrometry to analyze biotinylation patterns and identify modified peptides.
• Limited proteolytic digestion to assess changes in protein accessibility upon DNA binding.

Main Results:

• DNA binding reduced or eliminated biotinylation at specific lysine sites on Ku70 and Ku80.
• Biotinylation patterns in the Ku70 C-terminal SAP domain suggested DNA-induced structural alterations.
• Proteolytic digests revealed altered accessibility of Ku70 and Ku80 C-terminal domains upon DNA binding, with a specific 10 kDa peptide preferentially generated from non-DNA-bound Ku70.

Conclusions:

• DNA binding induces significant structural changes and domain movements in the Ku70 and Ku80 heterodimer.
• These DNA-dependent structural alterations in Ku are likely critical for the subsequent binding and activation of DNA-PKcs.
• This study provides the first evidence of DNA-induced structural modifications in Ku, offering a framework for understanding DNA-PK activation.