Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Breast Cancer type 2 susceptibility protein (BRCA2) is critical for maintaining genome stability through homologous recombination (HR) and replication fork protection.
• Proper nuclear localization and interaction dynamics are essential for BRCA2 function.
• The roles of specific domains, particularly the DNA-binding domain (DBD) and C-terminal domain (CTD), in BRCA2's cellular activities remain incompletely understood.

Purpose of the Study:

• To investigate the functional significance of the conserved DNA-binding domain (DBD) and C-terminal domain (CTD) of BRCA2.
• To define the contribution of these domains to HR function and nuclear localization dynamics.
• To explore how binding partners influence BRCA2's molecular architecture and its impact on cellular function.

Main Methods:

• Generation of mouse embryonic stem (ES) cells expressing BRCA2 variants lacking the DBD and/or CTD.
• Single-particle tracking to analyze the dynamic mobility of BRCA2 in the nucleus.
• Scanning force microscopy to assess structural changes in purified BRCA2 proteins upon binding partner interaction.

Main Results:

• BRCA2 mobility and its immobilization upon DNA damage were not significantly affected by C-terminal deletions.
• BRCA2 variants lacking the CTD and/or DBD exhibited defects in structural rearrangements.
• These structural defects correlated with impaired homologous recombination function in cells, suggesting roles beyond RAD51 delivery.

Conclusions:

• The DBD and CTD of BRCA2 are essential for its structural integrity and functional activity in homologous recombination.
• BRCA2's role in DNA damage response involves critical architectural changes influenced by its domains and binding partners.
• These findings highlight the importance of BRCA2's intrinsic structural properties for its multifaceted genome maintenance functions.