The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks
- Creators
- Lee, Joon
- Dunphy, William G.
Abstract
The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIβ-binding protein 1 (TopBP1), Rad17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both Rad17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, Rad17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, Rad17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure–function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.
Additional Information
© 2013 Lee and Dunphy. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). Received: Jan 10, 2013; Revised: Feb 21, 2013; Accepted: Feb 25, 2013. Published online before print March 6, 2013. We are grateful to laboratory members for helpful advice and comments on the manuscript. This work was supported by NIH grants GM070891 and GM043974 to W.G.D.Attached Files
Published - Mol._Biol._Cell-2013-Lee-1343-53.pdf
Supplemental Material - CombinedSupMats.pdf
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Additional details
- PMCID
- PMC3639046
- Eprint ID
- 39633
- Resolver ID
- CaltechAUTHORS:20130729-141626502
- GM070891
- NIH
- GM043974
- NIH
- Created
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2013-07-29Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field