NBN Phosphorylation regulates the accumulation of MRN and ATM at sites of DNA Double-Strand Breaks

Jie Wen; Karen Cerosaletti; Katherine J Schultz; Jocyndra A Wright; Patrick Concannon

doi:10.1038/onc.2012.443

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NBN Phosphorylation regulates the accumulation of MRN and ATM at sites of DNA Double-Strand Breaks

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NBN Phosphorylation regulates the accumulation of MRN and ATM at sites of DNA Double-Strand Breaks

Jie Wen, Karen Cerosaletti, Katherine J Schultz, Jocyndra A Wright and Patrick Concannon

Oncogene, Vol.32(37), pp.4448-4456

09/12/2013

DOI: 10.1038/onc.2012.443

PMCID: PMC3951136

PMID: 23146902

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Abstract

In response to ionizing radiation, the MRE11/RAD50/NBN (MRN) complex re-distributes to the sites of DNA double strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I- Ppo I, into human cells to generate site-specific DSBs. Direct transduction of I- Ppo I protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I- Ppo I into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine to alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine to glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared to control cells while blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.

ATM

DNA double-strand breaks

I-PpoI

MRN

nibrin (NBN)

Details

Title: Subtitle: NBN Phosphorylation regulates the accumulation of MRN and ATM at sites of DNA Double-Strand Breaks
Creators: Jie Wen - Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, United States, 22908
Karen Cerosaletti - Translational Research Program, Benaroya Research Institute, 1201 Ninth Avenue, Seattle, WA, 98101
Katherine J Schultz - University of Iowa, Stead Family Department of Pediatrics
Jocyndra A Wright - Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, United States, 22908
Patrick Concannon - Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, United States, 22908
Resource Type: Journal article
Publication Details: Oncogene, Vol.32(37), pp.4448-4456
DOI: 10.1038/onc.2012.443
PMID: 23146902
PMCID: PMC3951136
NLM abbreviation: Oncogene
ISSN: 0950-9232
eISSN: 1476-5594
Language: English
Date published: 09/12/2013
Academic Unit: Stead Family Department of Pediatrics
Record Identifier: 9984129301902771

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