Advertisement

Source Data

Transparent Process

Mec1/ATR regulates the generation of single‐stranded DNA that attenuates Tel1/ATM signaling at DNA ends

Michela Clerici, Camilla Trovesi, Alessandro Galbiati, Giovanna Lucchini, Maria Pia Longhese

Author Affiliations

  1. Michela Clerici1,,
  2. Camilla Trovesi1,,
  3. Alessandro Galbiati1,
  4. Giovanna Lucchini1 and
  5. Maria Pia Longhese*,1
  1. 1Dipartimento di Biotecnologie e Bioscienze, Università di Milano‐Bicocca, Milan, Italy
  1. *Corresponding author. Tel: 0039 0264483425; Fax: 0039 0264483565; E‐mail: mariapia.longhese{at}unimib.it
  1. These two authors contributed equally to the work.

  2. Conceived and designed the experiments: MC, CT, MPL. Performed the experiments: MC, CT, AG. Analysed the data: MC, CT, GL, MPL. Wrote the paper: MPL, GL.

View Full Text

Abstract

Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double‐strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA‐coated single‐stranded DNA (ssDNA), which arises from 5′–3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1‐ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53‐dependent and Rad53‐independent mechanisms. The mec1‐ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.

Synopsis

Embedded Image

Persistent Tel1 activation upon defective Mec1‐dependent resection in yeast interferes with checkpoint adaptation, exemplifying a role for the ATM/ATR switch previously observed in human cells.

  • A Mec1‐ad mutant variant that impaired resection of DNA ends was identified by searching for mec1 mutants failing to turn off the checkpoint after generation of a single irreparable DNA double‐strand break (DSB).

  • Mec1‐ad affects DSB resection by increasing the efficiency of γH2A generation, which in turn causes enhanced amount/persistence of the resection inhibitor Rad9 at DNA ends.

  • Defective DSB resection caused by either Rad9 excess or dysfunctions of the resection machinery leads to persistent Tel1‐dependent checkpoint signaling by prolonging MRX occupancy at DSBs.

  • Switch from Tel1‐ to Mec1‐dependent signaling activity promoted by DSB resection ensures proper termination of the checkpoint response.

Footnotes

  • The authors declare that they have no conflict of interest.

  • Received June 20, 2013.
  • Revision received October 24, 2013.
  • Accepted October 31, 2013.
View Full Text

Subscribers, please sign in with your username and password.

List of OpenAthens registered sites, including contact details.