Inhibition of MRN activity by a telomere protein motif

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles’ heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well

Binding of the TRF2 iDDR motif to RAD50 highlights a convergent evolutionary strategy to inactivate MRN at telomeres

Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which plays a critical role in the processing of double-stranded DNA breaks (DSBs). MRN orchestrates activation of the ATM kinase in the cellular DNA damage response (DDR), promotes DNA end-tethering aiding the nonhomologous end joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A previously identified protein motif (MIN, for MRN inhibitor) downregulates MRN activity via binding to RAD50 and has independently arisen at least twice, through convergent evolution of telomeric proteins Rif2 and Taz1, in budding and fission yeast respectively. We now provide a third example of convergent evolution for this binding mechanism for MRN at telomeres, by demonstrating that the iDDR motif of the human shelterin protein TRF2 binds to human RAD50 at the same site engaged by the MIN motif in the yeast proteins, despite lacking sequence homology. Modelling for the human CtIP interaction with RAD50 (necessary for activation of MRE11), and for the budding and fission yeast counterparts Sae2 and Ctp1, indicates that the interaction is mutually exclusive with binding of the iDDR/MIN motifs, pointing to a conserved mechanism for inhibition of MRN nuclease activity at telomeres

Binding of the TRF2 iDDR motif to RAD50 highlights a convergent evolutionary strategy to inactivate MRN at telomeres

Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which processes double-stranded DNA breaks (DSBs) via activation of the ATM kinase, promotes DNA end-tethering aiding the non-homologous end-joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A protein motif (MIN, for MRN inhibitor) inhibits MRN at budding yeast telomeres by binding to RAD50 and evolved at least twice, in unrelated telomeric proteins Rif2 and Taz1. We identify the iDDR motif of human shelterin protein TRF2 as a third example of convergent evolution for this telomeric mechanism for binding MRN, despite the iDDR lacking sequence homology to the MIN motif. CtIP is required for activation of MRE11 nuclease action, and we provide evidence for binding of a short C-terminal region of CtIP to a RAD50 interface that partly overlaps with the iDDR binding site, indicating that the interaction is mutually exclusive. In addition, we show that the iDDR impairs the DNA binding activity of RAD50. These results highlight direct inhibition of MRN action as a crucial role of telomeric proteins across organisms and point to multiple mechanisms enforced by the iDDR to disable the many activities of the MRN complex

Data for 'Binding of the TRF2 iDDR motif for RAD50 highlights a convergent evolutionary strategy to inactivate MRN at telomeres'

Data for paper published in Nucleic Acids Research (2024)Deposited data represent models generated by ColabFold in Protein Data Bank Format (.pdb). Software that can read molecular coordinate files in PDB format should allow visualisation of each model.Molecular Graphics Software:https://www.rcsb.org/docs/additional-resources/molecular-graphics-softwareColabFold / Alphafold2 models for telomeric proteins interacting with the head domain of Rad50Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which processes double-stranded DNA breaks (DSBs) via activation of the ATM kinase, promotes DNA end-tethering aiding the non-homologous end-joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A protein motif (MIN, for MRN inhibitor) evolved at least twice in yeast, in unrelated telomeric proteins Rif2 and Taz1, and inhibits MRN at telomeres by binding to RAD50. We identify the iDDR motif of human shelterin protein TRF2 as a third example of convergent evolution for this binding mechanism for MRN at telomeres, despite the iDDR lacking sequence homology to the MIN motif. CtIP is required for activation of MRE11 nuclease action, and we provide evidence for binding of a short C-terminal region of CtIP to a RAD50 interface that partly overlaps with the iDDR binding site, indicating that the interaction is mutually exclusive. In addition, we show that the iDDR impairs the DNA binding activity of RAD50. These results highlight direct inhibition of MRN action as a crucial role of telomeric proteins across organisms and point to multiple mechanisms enforced by the iDDR to disable the many activities of the MRN complex.</p