Xenopus ATR is a replication-dependent chromatin-binding protein required for the DMA replication checkpoint

Background: The DNA replication checkpoint ensures that mitosis is not init iated before DNA synthesis is completed. Recent studies using Xenopus extra cts have demonstrated that activation of the replication checkpoint and pho sphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DN A polymerase cc, and it has been suggested that the ATR kinase - so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase - may be an upstream component of t his response. It has been difficult to test this hypothesis as an ATR-defic ient system suitable for biochemical studies has not been available. Results: We have cloned the Xenopus laevis homolog of ATR (XATR) and studie d the function of the protein in Xenopus egg extracts. Using a chromatin-bi nding assay, we found that ATR associates with chromatin after initiation o f replication, dissociates from chromatin upon completion of replication, a nd accumulates in the presence of aphidicolin, an inhibitor of DNA replicat ion. Its association with chromatin was inhibited by treatment with actinom ycin D, an inhibitor of RNA primase. There was an early rise in the activit y of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon de pletion of ATR was accompanied by the loss of Chk1 phosphorylation. Conclusions: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymer ase ct. Depletion of ATR leads to premature mitosis in the presence and abs ence of aphidicolin, indicating that ATR is required for the DNA replicatio n checkpoint.