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Updated: May 11, 2025
Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
Published on: April 29, 2010
ATM priming and end resection-coupled phosphorylation of MRE11 is important for fork protection and replication
Huimin Zhang1, Youhang Li1,2, Sameer Bikram Shah1
1Department of Molecular and Cellular Biology, The Scripps Research Institute, La Jolla, CA 92037.
Abstract:
The MRE11/RAD50/NBS1 (MRN) complex plays multiple roles in the maintenance of genome stability. MRN is associated with replication forks to preserve fork integrity and is also required for end resection at double-strand breaks (DSBs) to facilitate homologous recombination (HR). The critical need for proper control of the MRE11 nuclease activity is highlighted by the extensive nascent strand DNA degradation driven by MRE11 in BRCA-deficient cells, leading to genome instability and increased sensitivity to chemotherapeutics. In this study, we identified a tightly controlled mechanism, elicited by sequential phosphorylation of MRE11 by ATM and ATR to regulate MRE11 nuclease activities through its DNA binding. Specifically, at DSBs, MRE11 phosphorylation by ATM at the C-terminal S676/S678 primes it for subsequent phosphorylation by ATR, whose activation is triggered by end resection which requires the MRE11 nuclease activity. This ATR-mediated phosphorylation in turn induces MRE11 dissociation from DNA, providing a feedback mechanism to regulate the extent of end resection. At stalled replication forks, however, without ATM priming, MRN is stably associated with forks despite ATR activation. Furthermore, the ATR phosphorylation-defective MRE11 mutants are retained at single-ended DSBs formed by fork reversal upon replication stress, leading to extensive degradation of nascent DNA strands. Importantly, this end resection-coupled MRE11 phosphorylation elicits another critical layer of fork protection of nascent DNA in addition to BRCA2, ensuring proper end resection that is sufficient for replication restart at reversed forks while maintaining fork stability.
Insights
The MRE11/RAD50/NBS1 (MRN) complex controls DNA repair. Sequential ATM and ATR phosphorylation of MRE11 regulates its nuclease activity, preventing excessive DNA degradation and maintaining genome stability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The MRE11/RAD50/NBS1 (MRN) complex is crucial for maintaining genome stability.
- MRN participates in replication fork integrity and DNA double-strand break (DSB) end resection for homologous recombination (HR).
- Uncontrolled MRE11 nuclease activity, particularly in BRCA-deficient cells, causes DNA degradation, genome instability, and sensitivity to chemotherapy.
Purpose of the Study:
- To elucidate the regulatory mechanism controlling MRE11 nuclease activity.
- To investigate the roles of ATM and ATR kinases in MRE11 regulation at DSBs and stalled replication forks.
- To understand how MRE11 phosphorylation impacts DNA repair and replication fork stability.
Main Methods:
- Investigated MRE11 phosphorylation by ATM and ATR in response to DNA damage.
- Utilized MRE11 phosphorylation-defective mutants to assess MRE11 function.
- Analyzed MRE11 association with DNA and nascent DNA strand degradation at replication forks and DSBs.
Main Results:
- Identified sequential ATM and ATR phosphorylation of MRE11 as a key regulatory mechanism.
- ATM phosphorylation at C-terminal sites primes MRE11 for ATR phosphorylation at DSBs, promoting MRE11 dissociation from DNA and limiting end resection.
- At stalled replication forks without ATM priming, MRN remains associated, and ATR-defective mutants show excessive nascent strand degradation.
Conclusions:
- Sequential MRE11 phosphorylation by ATM and ATR provides feedback control over end resection at DSBs.
- This phosphorylation mechanism is essential for protecting nascent DNA strands at reversed replication forks, ensuring replication restart and fork stability.
- MRE11 phosphorylation represents a critical layer of fork protection, complementing BRCA2 function.
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