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Two distinct modes of ATR activation orchestrated by Rad17 and Nbs1

Bunsyo Shiotani1, Hai Dang Nguyen, Pelle Håkansson

  • 1Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA. bshiotan@hiroshima-u.ac.jp

Cell Reports
|May 21, 2013
PubMed

Insights

The ATM- and Rad3-related (ATR) kinase regulates DNA damage response. This study reveals ATR phosphorylates RPA32 via Nbs1, crucial for repairing collapsed replication forks.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Genetics

Background:

  • The ATM- and Rad3-related (ATR) kinase is a key regulator of the DNA damage response.
  • Mechanisms of ATR activation and substrate specificity are not fully understood.

Purpose of the Study:

  • To elucidate the distinct mechanisms by which ATR phosphorylates Chk1 and RPA32 at replication-associated DNA double-stranded breaks (DSBs).
  • To investigate the role of Nbs1 in ATR-mediated RPA32 phosphorylation and its impact on replication fork stability.

Main Methods:

  • Investigated ATR substrate phosphorylation at DSBs using in vivo and in vitro assays.
  • Utilized Nbs1 mutants to dissect its role in ATR activation and RPA32 phosphorylation.
  • Assessed replication fork recovery in cells expressing Nbs1 mutants.

Main Results:

  • ATR phosphorylates Chk1 rapidly, while RPA32 phosphorylation at Ser33 is progressive and occurs during DSB resection.
  • RPA32 Ser33 phosphorylation relies on ATR and TopBP1 but is independent of Rad17 and dependent on Nbs1.
  • Nbs1's role in RPA32 phosphorylation is separable from ATM activation and DSB resection, depending on Nbs1-RPA interaction.

Conclusions:

  • ATR utilizes distinct pathways for Chk1 and RPA32 phosphorylation at replication-associated DSBs.
  • Nbs1-mediated ATR activation, dependent on RPA binding, is critical for repairing collapsed replication forks.

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