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  • Original Paper
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ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

Abstract

ATR is a large, >300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and purified the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, double-stranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we find that ATR has a substrate specificity similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we find that the kinase activity of ATR in the presence and absence of DNA is suppressed by caffeine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

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Acknowledgements

Recombinant human p53 (Hupp et al., 1992) and mdm2 were kind gifts from C Blattner and D Lane (Department of Biochemistry, University of Dundee), mutant S15A human p53 from B Hann (Onyx Corp.) and recombinant Replication Factor A (RPA) from J Russell (Department of Biochemistry, University of Dundee). cDNA encoding full length ATR was obtained from T Carr (MCR Cell Mutation Unit, University of Sussex), and mutant ATR and 4779 antibodies were from M Hoekstra and K Keegan (ICOS Corp). This work was supported by The Association for International Cancer Research and the MRC (to C Smythe.).

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Hall-Jackson, C., Cross, D., Morrice, N. et al. ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene 18, 6707–6713 (1999). https://doi.org/10.1038/sj.onc.1203077

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