Shortage of dNTPs underlies altered replication dynamics and DNA breakage in the absence of the APC/C cofactor Cdh1
J Garzón1, R Rodríguez1, Z Kong2
1Instituto de Biología Funcional y Genómica (IBFG)-CSIC, Salamanca University, Salamanca, Spain.
Abstract:
The APC/C-Cdh1 ubiquitin-ligase complex targets cell cycle regulators for proteosomal degradation and helps prevent tumor development and accumulation of chromosomal aberrations. Replication stress has been proposed to be the main driver of genomic instability in the absence of Cdh1, but the real contribution of APC/C-Cdh1 to efficient replication, especially in normal cells, remains unclear. Here we show that, in primary MEFs, acute depletion or permanent ablation of Cdh1 slowed down replication fork movement and increased origin activity. Partial inhibition of origin firing does not accelerate replication forks, suggesting that fork progression is intrinsically limited in the absence of Cdh1. Moreover, exogenous supply of nucleotide precursors, or ectopic overexpression of RRM2, the regulatory subunit of Ribonucleotide Reductase, restore replication efficiency, indicating that dNTP availability could be impaired upon Cdh1 loss. Indeed, we found reduced dNTP levels in Cdh1-deficient MEFs. Importantly, DNA breakage is also significantly alleviated by increasing intracellular dNTP pools, strongly suggesting that genomic instability is the result of aberrant replication. These observations highlight the relevance of APC/C-Cdh1 activity during G1 to ensure an adequate supply of dNTPs to the replisome, prevent replication stress and the resulting chromosomal breaks and, ultimately, suppress tumorigenesis.
Insights
The APC/C-Cdh1 complex ensures sufficient nucleotide supply for DNA replication. Its absence impairs replication fork speed and increases DNA breaks, highlighting its role in preventing genomic instability and cancer.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The anaphase-promoting complex/cyclosome (APC/C)-Cdc20 homolog 1 (Cdh1) complex degrades cell cycle regulators, preventing tumor formation and chromosomal instability.
- Replication stress is thought to cause genomic instability when Cdh1 is absent, but its role in normal cell replication is unclear.
Purpose of the Study:
- To investigate the role of APC/C-Cdh1 in DNA replication efficiency and its contribution to genomic stability in primary cells.
- To determine if dNTP availability is affected by Cdh1 deficiency and if this contributes to replication stress.
Main Methods:
- Acute depletion and permanent ablation of Cdh1 in primary mouse embryonic fibroblasts (MEFs).
- Analysis of replication fork movement and origin activity.
- Assessment of dNTP levels and DNA breakage.
- Manipulation of dNTP pools via nucleotide precursor supply or RRM2 overexpression.
Main Results:
- Cdh1 deficiency slowed replication fork progression and increased origin firing.
- Restoring dNTP levels via precursors or RRM2 overexpression improved replication efficiency.
- Reduced dNTP levels were observed in Cdh1-deficient MEFs.
- Increased dNTP pools alleviated DNA breakage, linking genomic instability to aberrant replication.
Conclusions:
- APC/C-Cdh1 activity in G1 is crucial for maintaining adequate dNTP supply for the replisome.
- Loss of Cdh1 leads to replication stress, DNA breakage, and genomic instability due to impaired dNTP availability.
- APC/C-Cdh1 function is vital for preventing tumorigenesis by ensuring replication fidelity.
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