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

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.

Heme deficiency sensitizes yeast cells to oxidative stress induced by hydroxyurea

“…To understand the molecular mechanism by which the checkpoint signaling is initiated at the replication forks (13,14), we have been searching for new DRC mutants. Because the fission yeast Schizosaccharomyces pombe is an established model for studying the cellular mechanisms that are conserved in humans, we carried out a genetic screen looking for new S. pombe mutants that are sensitive to the replication stress induced by HU.…”

“…To understand the initiation process of the DRC signaling at perturbed replication forks (13,14), we carried out a hus (HUsensitive) screen in S. pombe (16) to identify new mutants that are sensitive to the replication stress induced by HU. After removal of the mutants with known mutations that are sensitive to HU by extensive genetic crossing, the newly screened hus mutants were transformed with S. pombe genomic DNA expression libraries carrying the ura4 marker.…”

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

“…Similar to the DRC signaling in mammalian cells, Cds1 is activated by the sensor kinase Rad3 (ATR/Mec1) and stimulates most of the protective cellular responses in S. pombe. Although the activation mechanisms of the mediator kinases Cds1 (Xu et al 2006;Cai et al 2009;Xu and Kelly 2009) and CHK1 (Chen et al 2000;Han et al 2016) have been well characterized, the mechanism by which the sensor kinases are activated under replication stress remains incompletely understood (Bandhu et al 2014;Yue et al 2014). Because fission yeast is an established model for studying the cellular mechanisms that are conserved in higher eukaryotes, we have been addressing this issue by searching for new S. pombe mutants that are sensitive to the replication stress induced by hydroxyurea (HU).…”

Mechanism of regulation of ‘chromosome kissing’ induced by Fob1 and its physiological significance

“…Although it is known that an RPA complex is needed for Rad51/Rad52-mediated homologous recombination (Sung et al 2003), the recombination event requires RPA2-catalyzed steps that follow rather than precede the Fob1-mediated synaptic interaction. RPA is also involved in a checkpoint response by and loading of Mec1 at a single-stranded region using the adapter protein Ddc2 (Bandhu et al 2014). However, the latter is provoked by forkstalling caused by genotoxic stress.…”