Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

Abstract

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00474-13.

ACKNOWLEDGMENTS

We thank Rodney Rothstein, Hannah Klein, Munira Basrai, and Noel Lowndes for reagents.

This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (NHMRC) and in part by the Victorian Government's Operational Infrastructure Support Program (to J.H.), grant EX95-9508NI from the National Health Research Institute (NHRI), an Academia Sinica Investigator Award (to M.-D.T.), AIRC (IG grant no. 10343, to A.P.), and the Swedish Foundation for Strategic Research, the Swedish Research Council, and the Swedish Cancer Society (to A.C.).

N.C.H. performed most of the experiments; E.S.-W.C., S.-C.W., and M.-D.T. provided mass spectrometry results; R.B. and A.C. provided dNTP measurements; A.F. helped with tetrad dissections; A.P. provided reagents; N.C.H., E.S.-W.C., R.B., A.H., A.P., A.C., M.-D.T., and J.H. analyzed, interpreted, and discussed data; and N.C.H. and J.H. wrote the article.

Alessandro Fazio was on exchange from the Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.

We declare that we have no conflict of interest.