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MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks

Katrina Montales1, Kenna Ruis1, Howard Lindsay2

  • 1Department of Biological Sciences, Molecular and Computational Biology Section, University of Southern California, Los Angeles, California, United States of America.

Plos One
|August 2, 2022
PubMed

Insights

Topoisomerase II-binding protein (TOPBP1) transiently recruits to DNA double-strand breaks (DSBs) to activate Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase. Its recruitment involves MRN-dependent and independent pathways, but not the 9-1-1 complex or MDC1.

Area of Science:

  • DNA damage response
  • Cellular signaling pathways
  • Molecular biology

Background:

  • Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase activation requires specific activators like topoisomerase II-binding protein (TOPBP1) at DNA damage sites.
  • Understanding the recruitment dynamics of TOPBP1 to DNA double-strand breaks (DSBs) is crucial for initiating ATR signaling.

Purpose of the Study:

  • To investigate the recruitment mechanism of TOPBP1 to DSBs within the Xenopus egg extract (DMAX) system.
  • To elucidate the roles of the MRE11-RAD50-NBS1 (MRN) complex, CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) in TOPBP1 recruitment.

Main Methods:

  • Utilized the DSB-mediated ATR activation in Xenopus egg extract (DMAX) system.
  • Examined the recruitment and retention of TOPBP1 at DSBs using protein depletion and kinase inhibition strategies.
  • Assessed the impact of MRN, CtIP, and ATM on TOPBP1 localization to DSBs.

Main Results:

  • TOPBP1 exhibits transient presence at DSBs with a half-life under 10 minutes.
  • Loss of MRN partially inhibits TOPBP1 recruitment, while loss of CtIP has no effect.
  • ATM kinase activity inhibition enhances MRN and TOPBP1 recruitment, but prevents CtIP recruitment.
  • The 9-1-1 complex and MDC1 are not required for TOPBP1 recruitment to DSBs.

Conclusions:

  • TOPBP1 recruitment to DSBs for ATR activation occurs through both MRN-dependent and independent pathways.
  • ATM kinase activity modulates the recruitment of MRN, CtIP, and TOPBP1 to DSBs.
  • The 9-1-1 complex and MDC1 play dispensable roles in TOPBP1 recruitment to DSBs.

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