A dominant-negative MEC3 mutant uncovers new functions for the Rad17 complex and Tel1

Abstract

The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G 1 and G2 and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that has an unexpected phenotype. It inactivates the G1 checkpoint while it leaves the G2 response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-bystep the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G 1 cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G 2 checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.

Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint

“…Rad53 activation requires prior hyperphosphorylation of Rad9. This event is Mec1 dependent and can be observed in response to any kind of damage, both in G1 and G2, where Rad9 is already modified at a basal level (Emili, 1998; Sun et al , 1998; Vialard et al , 1998; Giannattasio et al , 2002). We compared the status of Rad9 after 4NQO treatment in wt and in cells lacking RAD14 or RAD2 , a gene coding for a factor of the structure‐specific endonuclease.…”

“…It is still unclear how Mec1 and its substrates are brought in close proximity onto DNA after genotoxic treatment. It has been recently shown that Mec1–Ddc2 and the PCNA‐like complex can be independently recruited onto damaged DNA (Kondo et al , 2001; Melo et al , 2001; Zou et al , 2002), and it has been suggested that the PCNA‐like complex might be involved in recruiting different substrates for Mec1 (Giannattasio et al , 2002; Zou et al , 2002). Critical Mec1 targets are Rad9 and Rad53; their modification allows these two proteins to interact, leading to Rad53 kinase activation.…”

The Unstructured C-Terminal Tail of the 9-1-1 Clamp Subunit Ddc1 Activates Mec1/ATR via Two Distinct Mechanisms

“…4). This residual response may operate through clamp-Tel1 interactions (Giannattasio et al, 2002). Finally, damage sensitivity measurements of the mutants lends additional support to a model that direct activation by 9-1-1 and indirect activation by 9-1-1 via Dpb11 contribute to checkpoint function in G2/M.…”

A Tel1/MRX-Dependent Checkpoint Inhibits the Metaphase-to-Anaphase Transition after UV Irradiation in the Absence of Mec1

“…In S. cerevisiae, Mec1 plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while Tel1 functions in the DNA damage response are inferred from the finding that its absence increases the sensitivity of mec1 mutants to genotoxic agents (62,84), it impairs the checkpoint response to phleomycin treatment during S phase (67), and it allows the G 2 /M transition after DNA damage in a dominant-negative MEC3 mutant (29). Moreover, altering DSB processing in mec1⌬ cells by deletion of SAE2 or non-null mutations in the RAD50 and MRE11 genes triggers Tel1/MRX-dependent Rad53 phosphorylation and interaction with Rad9 after MMS and hydroxyurea treatment, suggesting that accumulation of DNA lesions that specifically activate Tel1 might take place under these conditions (104).…”

“…Although TEL1 deletion by itself does not confer sensitivity to DNA-damaging agents, Tel1 functions in the DNA damage response are inferred from the finding that its absence increases the sensitivity of mec1 mutants to genotoxic agents (62,84), and it impairs the checkpoint response to phleomycin treatment during S phase (67). Tel1 is also responsible for the G 2 arrest after DNA damage in a dominant-negative MEC3 mutant (29). Moreover, TEL1 overexpression can suppress both cell lethality and hypersensitivity to DNA-damaging agents of mec1⌬ and ddc2⌬ mutants, indicating that excess Tel1 can bypass the requirements for the Mec1-Ddc2 complex (11,84).…”