Human TopBP1 localization to the mitotic centrosome mediates mitotic progression

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• TopBP1, a protein with BRCA1 C-terminal (BRCT) domains, is vital for DNA damage response and replication during interphase.
• During mitosis, TopBP1 localizes to centrosomes, essential for sister chromatid separation.
• This centrosome localization is mediated by the TopBP1 C-terminal region (TbpCtr).

Purpose of the Study:

• To investigate the role of TopBP1 centrosome localization in mitotic progression.
• To identify the specific region and residues responsible for TopBP1's mitotic centrosome targeting.
• To determine the functional consequences of disrupting TopBP1 centrosome localization.

Main Methods:

• Utilized GST and DsRed2 tags fused to the TbpCtr to assess its centrosome localization.
• Introduced mutations at Ser 1273 and Lys 1317 within TbpCtr to evaluate their impact on localization.
• Employed ectopic expression of TbpCtr and its mutants to displace endogenous TopBP1 from centrosomes.

Main Results:

• The TbpCtr (residues 1259-1420) functions as a mitosis-specific centrosome localization signal (CLS).
• Mutations at Ser 1273 and/or Lys 1317 impaired the CLS function.
• Disrupting TopBP1 centrosome localization by TbpCtr overexpression prolonged prometaphase and metaphase and reduced inter-kinetochore distances.

Conclusions:

• TopBP1's localization to mitotic centrosomes is mediated by its C-terminal region and specific residues.
• This localization is essential for the timely progression through mitosis.
• Disruption of TopBP1 centrosome targeting impacts key mitotic events while preserving the spindle assembly checkpoint.