ATRMec1 phosphorylation-independent activation of Chk1 in vivo

Abstract

The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks. The current model is that after DNA damage or replication blocks, ATRMec1 phosphorylates Chkl on the non-catalytic C-terminal domain. However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus in vivo remains largely unknown. In this study we used an in vivo assay to examine the role of the C terminus of Chk1 in the response to DNA damage and replication blocks. The conserved ATRMec1 phosphorylation sites were essential for the checkpoint response to DNA damage and replication blocks in vivo; that is, that mutation of the sites caused lethality when DNA replication was stalled by hydroxyurea. Despite this, loss of the ATRMec1 phosphorylation sites did not change the kinase activity of Chk1 in vitro. Furthermore, a single amino acid substitution at an invariant leucine in a conserved domain of the non-catalytic C terminus restored viability to cells expressing the ATRMec1 phosphorylation site-mutated protein and relieved the requirement of an upstream mediator for Chk1 activation. Our findings show that a single amino acid substitution in the C terminus, which could lead to an allosteric change in Chk1, allows it to bypass the requirement of the conserved ATRMec1 phosphorylation sites for checkpoint function. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.