Abstract
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.
Highlights
The DNA damage response pathways coordinate DNA repair, replication and cell-cycle progression to maintain genome integrity [1,2]
We examined whether Mec1 catalytic activity is increased after DNA damage
Cells expressing HA-tagged Mec1 (Mec1-HA) protein were arrested with nocodazole and treated with methylmethane sulfonate (MMS)
Summary
The DNA damage response pathways coordinate DNA repair, replication and cell-cycle progression to maintain genome integrity [1,2]. Initiation of DNA damage responses requires two evolutionarily conserved phosphoinositide 3-kinase (PI3K)-related protein kinases: ATM and ATR. In the budding yeast Saccharomyces cerevisiae, Mec, the ATR ortholog, plays a critical role in the DNA damage response throughout the cell-cycle [1,4]. Mec forms a stable complex with Ddc (ATRIP in human) [5,6,7], which recruits Mec to sites of DNA damage by interacting with replication protein A (RPA)-coated ssDNA [8,9,10,11]. Phosphorylated histone H2A creates a DNA damage mark similar to phosphorylated histone H2AX (cH2AX) in human [4,13]. Mec phosphorylates Rad at sites of DNA damage [16,17]. The Rad9Rad interaction promotes Rad autophosphorylation and allows Mec to phosphorylate Rad, leading to hyperphosphorylation and activation of Rad53 [20,21,22,23]
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