Tel2 Is Required for Activation of the Mrc1-mediated Replication Checkpoint

Abstract

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2⌬ strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1⌬ cells especially at S phase. In contrast, the cds1⌬swi1⌬ mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.

A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability

“…The TEL2/ CLK2 orthologs in C. elegans and the budding yeast S. cerevisiae are important regulators of telomere length (Runge and Zakian 1996;Kota and Runge 1999;Benard et al 2001;Lim et al 2001). In mammals and the fission yeast Schizosaccharomyces pombe, TEL2/CLK2 has been heavily implicated in the ATR signaling pathway and the S-phase checkpoint (Collis et al 2007(Collis et al , 2008Shikata et al 2007;Danielsen et al 2009). The connection between TEL2 and the PIKK family helps explain the pleiotrophic nature of the phenotypes of TEL2 mutants in different organisms.…”

How ATR turns on: TopBP1 goes on ATRIP with ATR: Figure 1.

“…This study by Mordes et al (2008) has also reiterated the role of ATRIP in stabilizing the interaction between TopBP1 and ATR for ATR activation, illustrating the involvement of ATRIP in multiple steps of ATR regulation. However, some common regulation may exist among PIKKs, as suggested by the finding that the Tel2 protein can regulate the stability of all PIKK family members via interaction with conserved N-terminal HEAT repeats (Shikata et al 2007;Takai et al 2007). In some cases, these regulatory mechanisms can even functionally connect family members.…”

Differential regulation of the PIKK kinase family by Tel2

“…Subsequent studies in many organisms have demonstrated a conserved role for Tel2 in the DNA damage checkpoint response. [2][3][4][5] The absence of recognizable domains in Tel2 has been a major hindrance in understanding how it is able to operate at the molecular level. The discovery that S. pombe and mammalian Tel2 interact directly with all phosphatidylinositol 3-kinase related protein kinase (PIKK) family members (which include ATM, ATR, DNA-PKcs, mTOR, FRAP and SMG1) raised the possibility that Tel2 may act as a universal regulator of the PIKK family of kinases.…”

“…The discovery that S. pombe and mammalian Tel2 interact directly with all phosphatidylinositol 3-kinase related protein kinase (PIKK) family members (which include ATM, ATR, DNA-PKcs, mTOR, FRAP and SMG1) raised the possibility that Tel2 may act as a universal regulator of the PIKK family of kinases. [3][4][5] A recent study has shown that conditional loss of Tel2 in mouse embryonic fibroblast (MEF) cells results in the dramatic reduction in protein levels of all PIKK family members. 5 This finding led de Lange and colleagues to propose that Tel2 is a highly conserved regulator of PIKK stability.…”

“…However, the Tel2 equivalents in fission yeast, worms and mammalian cells interact with ATR (Yeast Mec1) and are essential for its function. [3][4][5] It is notable that these studies analyzed deletion, depletion or conditional alleles that eliminate the entire Tel2 protein. Where the current study differs is in the use of the nonessential allele of Tel2 (tel2-1), which expresses the entire Tel2 protein with a single point mutant at Ser129 that compromises the Tel1 and Mec1 interaction site.…”