Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

Abstract

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.

Regulation of Elg1 activity by phosphorylation

“…The sequence context of these serines, however, is incompatible with the CDK1 cell cycle kinase, which is known to regulate genome stability proteins in coordination with DDR kinases. [36][37][38] The DNA damage response (DDR) has been genetically dissected in the yeast S. cerevisie: The main control is carried out by the phosphatidyl-inositol 3-kinase-like kinase (PI3KK) Mec1, an ortholog of the human ATM and ATR kinases. Mec1 controls the downstream kinases Chk1 and Rad53 through the Rad9 and Mrc1 adaptor proteins, which amplify signals created by chromosomal damage (e.g., DSBs) and replication slowdown or stalling, respectively.…”

Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases

“…Another substrate of the S. cerevisiae Cdc28 is the Srs2 helicase, the phosphorylation of which prevents its sumoylation and targets it to dismantle specific DNA structures, such as D-loops, in a helicase-dependent manner during homologous recombination 61 . During DNA damage repair Cdc28 also phosphorylates the G2 checkpoint protein Rad9 62–64 and the resection nuclease Dna2 65 . Collectively, these studies present a strong case for the requirement of CDK activity in the resection of DSB-ends and in mediating the choice between the homologous recombination and NHEJ repair pathways in yeast 66 .…”

Mechanisms and regulation of DNA replication initiation in eukaryotes

“…Like DDK, S-CDK is required for replication (Broek et al, 1991, Hayles et al, 1994) and exists in an inactive conformation by engaging Sic1, a repressive protein that restricts S-CDK activity into late G1/S phase (Mendenhall, 1993, Donovan et al, 1994, Schwob et al, 1994, Knapp et al, 1996, Schneider et al, 1996). Redundant regulation of S-CDK occurs by the cell-cycle dependent expression of activating cyclins (Abreu et al, 2013). It is currently unclear whether there is a conserved sequential order for DDK and CDK activity.…”