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Function of the ATR N-terminal domain revealed by an ATM/ATR chimera

Xinping Chen1, Runxiang Zhao, Gloria G Glick

  • 1Department of Biochemistry, Vanderbilt University, Pierce Avenue, Nashville, TN 37232, USA.

Experimental Cell Research
|March 23, 2007
PubMed

Insights

Researchers fused a domain from ATR kinase to ATM kinase, enabling it to bind ATRIP and localize to DNA damage sites. However, the modified ATM kinase remained defective in checkpoint signaling, highlighting the complexity of DNA damage response pathways.

Area of Science:

  • Cellular biology
  • Molecular oncology
  • DNA damage response

Background:

  • ATM and ATR kinases are crucial for cell cycle checkpoint signaling.
  • They share substrate similarity but differ in DNA damage sensing mechanisms.
  • ATR interacts with ATRIP to sense single-stranded DNA, while ATM interacts with Nbs1 for double-strand breaks.

Purpose of the Study:

  • To investigate the role of the ATR N-terminus in ATRIP binding and localization.
  • To determine if ATRIP binding and localization can be conferred to ATM.
  • To assess the functional consequences of ATR N-terminus mediated ATRIP binding on ATM function.

Main Methods:

  • Protein engineering to create a fusion protein (ATM*) by attaching the ATR N-terminus to ATM.
  • Assessing ATRIP binding and ssDNA association of ATM*.
  • Evaluating ATM* localization to DNA damage sites (replication forks, double-strand breaks).
  • In vitro kinase assays and in vivo phosphorylation analysis of ATM*.
  • Complementation assays to test ATM* function in ATM- or ATR-deficient cells.

Main Results:

  • The ATR N-terminus was sufficient to bind ATRIP and RPA-coated ssDNA when fused to ATM.
  • The fusion protein (ATM*) efficiently localized to stalled replication forks and double-strand breaks.
  • ATM* exhibited normal in vitro kinase activity and in vivo phosphorylation.
  • Despite these properties, ATM* failed to complement cellular deficiencies in ATM or ATR signaling.

Conclusions:

  • The N-terminus of ATR is sufficient for ATRIP binding and localization to sites of replication stress.
  • ATRIP binding and localization to damage sites are necessary but not sufficient for ATM/ATR checkpoint signaling.
  • ATM and ATR signaling pathways involve more complex regulatory mechanisms beyond N-terminal interactions and localization.

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