Abstract
ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1ATM and Rad3ATR recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1ATM to telomeres independently of the previously established Nbs1 C-terminal Tel1ATM interaction domain. Recruitment of Tel1ATM to telomeres in nbs1-c60Δ cells, which lack the C-terminal 60 amino acid Tel1ATM interaction domain of Nbs1, was dependent on Rad3ATR-Rad26ATRIP, but the kinase domain of Rad3ATR was dispensable. Thus, our results establish that the Rad3ATR-Rad26ATRIP complex contributes to the recruitment of Tel1ATM independently of Rad3ATR kinase activity, by a mechanism redundant with the Tel1ATM interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3ATR-Rad26ATRIP to telomeres. In response to replication stress, mammalian ATR–ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR–ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity.
Highlights
ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related), members of the phosphoinositol-3-kinase like kinase (PIKK) family, are central players in coordinating cellular responses to various forms of DNA damage, such as DNA double-stranded breaks (DSBs) and problems that are encountered by DNA replication forks, in eukaryotic cells [1,2]
Our current study in fission yeast demonstrates that the previously defined C-terminal Tel1ATM interaction domain of the DNA repair protein Nbs1, which contributes to recruitment of Tel1ATM to DSBs, is dispensable for recruitment of Tel1ATM to telomeres, due to a previously unrecognized kinase-independent role of ATR in recruitment of Tel1ATM to telomeres
Since proteins responsible for proper maintenance of telomeres and cellular responses to DNA damage are highly conserved between fission yeast and mammalian cells, a newly uncovered molecular crosstalk between ATM and ATR might play critical roles in telomere maintenance and DNA damage responses in mammalian cells
Summary
ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related), members of the phosphoinositol-3-kinase like kinase (PIKK) family, are central players in coordinating cellular responses to various forms of DNA damage, such as DNA double-stranded breaks (DSBs) and problems that are encountered by DNA replication forks, in eukaryotic cells [1,2]. Previous studies have identified the Mre11-Rad50-Nbs (MRN) DNA repair complex as a key player in the activation of ATM kinase in response to DSBs [1,4]. The MRN complex interacts with ATM through an evolutionarily conserved C-terminal motif in its Nbs subunit, and this interaction is critical for recruitment of ATM to DSBs and phosphorylation of downstream targets by ATM [5,6]. ATRIP (ATR-Interacting Protein) interacts with ATR through its evolutionarily conserved extreme Cterminal motif and promotes recruitment of ATR to sites of DNA damage [6]. RPA (Replication Protein A)-coated singlestranded DNA (ssDNA) serves as a platform for recruitment of the ATR-ATRIP complex, where phosphorylation of various downstream targets can take place [7]. Besides its role in activation of ATM, the MRN complex has been shown to contribute to ATR signaling in mammalian cells [8]
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