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A phosphorylation switch at MRE11 links ATM-ATR and calcium signaling to safeguard stalled replication fork stability
Weihang Valerie Chai1, Manobendro Ray1, Chih-Chun Chang2
1Rosalind Franklin University of Medicine and Sciences, Chicago Medical School.
Abstract:
MRE11 safeguards genome stability at stalled replication forks, where its activity must be tightly controlled to prevent nascent strand DNA degradation (NSD). However, the upstream signaling mechanisms that limit NSD remain poorly defined. Here, we identify Ser649 (S649) as a previously unrecognized phosphorylation site that limits MRE11 association with stalled forks. We show that S649 phosphorylation is robustly induced by replication stress or elevated cytosolic calcium levels, and is mediated by the calcium-responsive CaMKK2-AMPKα axis in concert with ATR, but independently of CHK1. Loss of S649 phosphorylation enhances MRE11 binding to DNA and increases its association with stalled forks, driving excessive NSD, elevated DNA damage, and increased sensitivity to PARP inhibition. We find that the ATM-mediated S676/S678 phosphorylation primes S649 phosphorylation, which in turn facilitates subsequent phosphorylation of SQ/TQ sites in MRE11. Moreover, we find that CaMKK2-AMPKα activation requires ATR but is independent of ATM. Collectively, our findings reveal a hierarchical signaling mechanism that couples calcium signaling with ATM/ATR pathways to prevent NSD at stalled forks and preserve genome integrity.
Insights
A newly discovered MRE11 phosphorylation site, Ser649, prevents DNA degradation at stalled replication forks. This calcium- and ATR-dependent signaling pathway is crucial for maintaining genome stability.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- MRE11 protein is essential for genome stability, particularly at stalled replication forks.
- Uncontrolled MRE11 activity can lead to nascent strand DNA degradation (NSD), compromising genomic integrity.
- The signaling pathways that regulate MRE11 activity at stalled forks are not fully understood.
Purpose of the Study:
- To identify novel regulatory mechanisms controlling MRE11 activity at stalled replication forks.
- To investigate the role of phosphorylation in limiting MRE11-mediated nascent strand DNA degradation (NSD).
Main Methods:
- Phosphorylation site mapping of MRE11.
- Replication stress induction and calcium signaling manipulation.
- Western blotting and immunoprecipitation assays.
- Analysis of MRE11 binding to DNA and stalled forks.
- Assessment of DNA damage and sensitivity to PARP inhibition.
Main Results:
- Serine 649 (S649) on MRE11 was identified as a key phosphorylation site limiting its association with stalled forks.
- S649 phosphorylation is induced by replication stress and elevated cytosolic calcium via the CaMKK2-AMPKα axis and ATR.
- Loss of S649 phosphorylation leads to increased MRE11 binding, excessive NSD, DNA damage, and sensitivity to PARP inhibitors.
- ATM-mediated phosphorylation of S676/S678 primes MRE11 for S649 phosphorylation.
Conclusions:
- A hierarchical signaling cascade involving calcium, CaMKK2-AMPKα, ATR, ATM, and MRE11 phosphorylation regulates MRE11 activity.
- This pathway prevents nascent strand DNA degradation at stalled replication forks, thereby preserving genome integrity.
- The findings reveal a critical mechanism coupling calcium signaling with DNA damage response pathways.
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