Shieldin complex assembly kinetics and DNA binding by SHLD3

Area of Science:

• Molecular biology
• DNA repair mechanisms
• Protein complex assembly

Background:

• The Shieldin complex is a key pro-non-homologous end joining (NHEJ) factor that represses end resection at DNA double-strand breaks (DSBs).
• The precise molecular mechanisms governing Shieldin complex assembly and its recruitment to DSBs remain largely unknown.
• The presence of two REV7 molecules within Shieldin, known for their ability to transition between native states, suggests a dynamic regulatory role in complex formation.

Purpose of the Study:

• To elucidate the molecular details of Shieldin complex assembly.
• To identify the rate-limiting step in Shieldin recruitment to DNA double-strand breaks.
• To understand the role of SHLD3 and REV7 interactions in Shieldin complex dynamics.

Main Methods:

• Identification and characterization of a DNA binding domain in SHLD3.
• Analysis of protein-protein interactions between SHLD3, SHLD2, and REV7.
• Kinetic studies to determine the rates of interaction between Shieldin components.

Main Results:

• A promiscuous DNA binding domain was identified in SHLD3.
• SHLD3 interacts with a dimer of REV7 molecules at its N-terminus.
• The interaction between SHLD3 and the first REV7 molecule is significantly slow, while subsequent interactions involving SHLD2 and a second REV7 are rapid and do not require structural changes.

Conclusions:

• The slow interaction between SHLD3 and REV7 represents the rate-limiting step in Shieldin complex assembly.
• These findings provide critical insights into the dynamic regulation of Shieldin complex formation and its recruitment to DNA double-strand breaks.
• Understanding these molecular details is essential for comprehending DNA repair pathways and their regulation.