Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism

Author Summary When DNA replication is blocked and DNA damage occurs, checkpoints arrest the cell cycle in eukaryotic cells, allowing DNA replication and repair to take place. The major regulators of the DNA damage checkpoint response are the phosphoinositide 3-kinase (PI3K)-related protein kinases, including ATM and ATR. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. ATM/Tel1 acts in response to double-strand breaks. By contrast, ATR/Mec1 recognizes many different types of DNA damage. Mec1 forms a complex with Ddc2 (ATRIP ortholog) that recruits Mec1 to sites of DNA damage. We isolated a ddc2 mutation that confers defects in DNA damage responses but does not impair Mec1 recruitment. We found that the catalytic activity of Mec1 increases in a Ddc2-dependent manner after DNA damage. Previous studies have demonstrated that Mec1 activation occurs through two independent pathways at G1 and G2/M: one pathway through Ddc1, a subunit of the checkpoint clamp and the second through Dpb11, the TopBP1 ortholog. We found that Mec1 activation occurs at least in part independently of Ddc1 and Dpb11. Our results suggest that Ddc2 stimulates Mec1 by a different mechanism than Ddc1 or Dpb11. The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.