ATR checkpoint kinase and CRL1βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

Chromosomal DNA replication is a highly conserved process in all eukaryotes that starts at specific sites along DNA known as replication origins. The activation of DNA replication origins requires assembly of prereplicative complexes (pre-RCs) followed by recruitment of additional initiating factors and formation of replisomes (Bell and Dutta 2002; Sclafani and Holzen 2007). Assembly of the pre-RCs onto each replication origin occurs during the early G1 phase of the cell cycle through sequential recruitment of the origin recognition complex (ORC), CDC6, CDT1, and minichromosome maintenance 2–7 (MCM2–7 complex) proteins. However, excess MCM2–7 complexes are loaded, leading to the licensing of excess origins, most of which are dormant and passively replicated in the normal S phase. The excess licensed origins, however, serve as backup origins that can complete DNA replication when existing forks stall (Ibarra et al. 2008; Ge and Blow 2010). In the presence of DNA-damaging agents, when newly fired replication forks stall after replicating a few hundred bases, many additional (formerly dormant) origins begin firing in mammalian cells (Ge et al. 2007; Courbet et al. 2008; Karnani and Dutta 2011). DNA replication is often the target of anti-cancer drugs, leading to the generation of stalled replication forks (Hoeijmakers 2001). ATR kinase recognizes these stalled replication forks and activates the intra-S-phase checkpoint signaling cascade. The activation of ATR requires the replication protein A (RPA) complex RPA70–RPA32–RPA14 and ATRIP (Cortez et al. 2001; Ball et al. 2005; Cimprich and Cortez 2008). The RPA complex coats ssDNA at stalled replication forks and recruits ATR and other checkpoint proteins (Zou and Elledge 2003). Once activated, ATR triggers the checkpoint by phosphorylating many downstream targets (including RPA, CHK1, and p53), ultimately leading to arrest of the cell cycle in S phase. Since transcription of neighboring genes is often repressed by even a single DNA double-strand break (DSB) in mammalian cells by active pathways using ATM and DNA-dependent protein kinases (Kruhlak et al. 2007; Shanbhag et al. 2010; Pankotai et al. 2012), we wondered whether and how stalled replication forks produced by anti-cancer drugs affect the transcription of neighboring genes. Doxorubicin (DOX) is a widely used cancer chemotherapeutic drug that works by intercalating into dsDNA, inhibiting the activity of DNA topoisomerase II (Bodley et al. 1989; Capranico et al. 1990). Despite the extensive use and study of this drug, the locations of replication origins that fire in the presence of DOX, the locations of stalled forks, and the effects of the stalled forks on neighboring gene expression have not been studied. Previously, we found that hydroxyurea (HU), a ribonucleotide reductase inhibitor (Elford 1968), activates clusters of dormant replication origins that produce clusters of forks that stall after a few hundred bases of DNA synthesis (Karnani and Dutta 2011). Many of these neoreplication origin clusters overlap with coding genes. We therefore hypothesized that chemotherapy drugs like DOX could activate similar clusters of neoreplication origins and produce clusters of stalled replication forks so that the cis effects of these stalled forks on the local transcription machinery would be part of the gene expression changes seen in cells treated with DOX. We show that DOX treatment produces clusters of stalled replication forks at specific sites in the genome and that the production of those clusters decreases the transcription of neighboring genes due to active changes in the chromatin and detachment of RNA polymerase II. Surprisingly, the transcriptional repression was not simply due to the mechanical effects of local clusters of stalled forks but an ATR-dependent checkpoint pathway that down-regulates ASF1a, a histone chaperone known to act in association with replication forks. The CRL1βTRCPE3 ligase complex polyubiquitinates ASF1a and targets it for degradation by proteasomes. This is a new mechanism by which a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks. These specific changes in gene expression produced by anti-S-phase chemotherapy drugs like DOX will contribute to the efficacy or toxicity of this class of drugs. Finally, decrease of ASF1a makes cancer cells more sensitive to DOX, suggesting that the homozygous deletion or underexpression of this histone chaperone, as seen in several cancers, could be a personalized tumor marker for sensitivity to DOX.