Shortage of dNTPs underlies altered replication dynamics and DNA breakage in the absence of the APC/C cofactor Cdh1

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.This research was funded by grants from the Spanish Ministry of Economy and Competitiveness MINECO (CSD2007-0015, BFU2011-28274 and BFU2014-55439) and Junta de Castilla y León (CSI151U13 and CSI084U16), the Swedish Cancer Society, the Knut and Alice Wallenberg Foundation and the Swedish Research Council (AC). IGH is supported by Fundación Científica de la Asociación Española contra el Cáncer (AECC). JG and RR were recipients of CSIC JAE and FPU predoctoral fellowships (MINECO).Peer reviewe