A tel2 Mutation That Destabilizes the Tel2-Tti1-Tti2 Complex Eliminates Rad3^ATR Kinase Signaling in the DNA Replication Checkpoint and Leads to Telomere Shortening in Fission Yeast

Abstract

In response to perturbed DNA replication, ATR (ataxia telangiectasia and Rad3-related) kinase is activated to initiate the checkpoint signaling necessary for maintaining genome integrity and cell survival. To better understand the signaling mechanism, we carried out a large-scale genetic screen in fission yeast looking for mutants with enhanced sensitivity to hydroxyurea. From a collection of ∼370 primary mutants, we found a few mutants in which Rad3 (ATR ortholog)-mediated phospho-signaling was significantly compromised. One such mutant carried an uncharacterized mutation in tel2, a gene encoding an essential and highly conserved eukaryotic protein. Previous studies in various biological models have shown that Tel2 mainly functions in Tel2-Tti1-Tti2 (TTT) complex that regulates the steady-state levels of all phosphatidylinositol 3-kinase-like protein kinases, including ATR. We show here that although the levels of Rad3 and Rad3-mediated phospho-signaling in DNA damage checkpoint were moderately reduced in the tel2 mutant, the phospho-signaling in the DNA replication checkpoint was almost completely eliminated. In addition, the tel2 mutation caused telomere shortening. Since the interactions of Tel2 with Tti1 and Tti2 were significantly weakened by the mutation, destabilization of the TTT complex likely contributes to the observed checkpoint and telomere defects.

The RecQ DNA helicase Rqh1 promotes Rad3ATRkinase signaling in the DNA replication checkpoint pathway of fission yeast

“…2B). Though more severe, the similar defects in the DRC and the DDC have also been described in the previously reported tel2-C307Y mutant (1).…”

“…The method for examining Rad3 phosphorylation of Mrc1-Thr 645 , Rad9-Thr 412 and Cds1-Thr 11 using the phosphor-specific antibodies has been described in our previously studies (1,12,43). The myc or HA tagged proteins was examined by Western blotting by using mouse monoclonal antibodies.…”

“…Studies in various eukaryotic organisms have shown that the primary cellular response to HU treatment is the activation of the DRC (3,17). We have previously reported one of the screened mutants tel2-C307Y that is highly sensitive to the replication stress induced by HU or DNA damage (1). Here we report our characterization of a group of rqh1 mutants.…”

“…To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome looking for mutants with defects in Rad3 kinase signaling. In addition to the previously reported tel2-C307Y mutant (1), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations except a start codon mutation are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint.…”

CLK-2/TEL2 is a conserved component of the nonsense-mediated mRNA decay pathway

“…CLK-2 (CLocK–biological timing–abnormality) impacts multiple cellular and developmental processes [ 21 – 23 ]. Its orthologs, from yeast to human, regulate telomere length and DNA damage response [ 24 – 27 ]. Additionally, biochemical studies demonstrated that the human counterpart of CLK-2, TELO2, functions as a scaffold for the assembly of a protein complex, the R2TP complex, which mediates the assembly of phosphatidylinositol 3-kinase-related kinases (PIKKs), such as ATM and ATR (critical for DNA damage response) and, importantly, the NMD kinase SMG1 [ 28 , 29 ].…”