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Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair.

Limbo O, Moiani D, Kertokalio A, Wyman C, Tainer JA, Russell P - Nucleic Acids Res. (2012)

Bottom Line: X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions.The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure.Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA.

ABSTRACT
The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.

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Mre11-W248R is proficient in Tel1 activation but partially defective for Ctp1 recruitment. (A) Immunoblot of phosphorylated histone H2A (γH2A) and total H2A with or without treatment with 90 Gy of IR. Graph represents γH2A expression relative to total H2A with error bars representing standard deviation from the mean of three independent experiments. (B) γH2A levels induced specifically by IR treatment determined by subtracting basal levels of γH2A from IR-treated shown in Panel A. (C) Southern blot probing for telomere-associated sequences (TAS1) from genomic DNA isolated from indicated genetic backgrounds and digested with EcoRI (top panel). Ethidium bromide (EtBr)-stained gel shows DNA loading (bottom panel). (D) Chromatin immunoprecipitation of Ctp1-TAP to an HO-induced DNA double-strand break in mre11+ and mre11-W248R backgrounds. Schematic of chromosome I containing HO break site and the relative distances and expected DNA sizes assayed by multiplex PCR is shown on top.
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gks954-F6: Mre11-W248R is proficient in Tel1 activation but partially defective for Ctp1 recruitment. (A) Immunoblot of phosphorylated histone H2A (γH2A) and total H2A with or without treatment with 90 Gy of IR. Graph represents γH2A expression relative to total H2A with error bars representing standard deviation from the mean of three independent experiments. (B) γH2A levels induced specifically by IR treatment determined by subtracting basal levels of γH2A from IR-treated shown in Panel A. (C) Southern blot probing for telomere-associated sequences (TAS1) from genomic DNA isolated from indicated genetic backgrounds and digested with EcoRI (top panel). Ethidium bromide (EtBr)-stained gel shows DNA loading (bottom panel). (D) Chromatin immunoprecipitation of Ctp1-TAP to an HO-induced DNA double-strand break in mre11+ and mre11-W248R backgrounds. Schematic of chromosome I containing HO break site and the relative distances and expected DNA sizes assayed by multiplex PCR is shown on top.

Mentions: Along with its functions in DNA repair, the MRN complex also recruits the checkpoint kinase ATMTel1 to DNA lesions through interactions with the C-terminus of Nbs1 (3,54,55). ATMTel1 activity therefore depends on the assembly of MRN complex and its capacity to bind DSBs. Thus, phosphorylation of ATMTel1 substrates provides an in vivo readout for these events. The best characterized substrate of Tel1 in fission yeast is histone H2A (analogous to H2AX in mammals), which is also phosphorylated by Rad3ATR (56,57). Therefore, Tel1 activity can be specifically assayed by performing experiments in a rad3Δ background. As expected, IR-induced formation of phospho-H2A (γH2A) was partially maintained in rad3Δ and tel1Δ single mutants but was abolished in the rad3Δ tel1Δ double mutant (Figure 6A and B). γH2A was absent in mre11Δ rad3Δ cells, showing that Mre11 is required for Tel1 activity at DSBs (3). Importantly, γH2A was formed in mre11-W248R rad3Δ cells (Figure 6A and B). Thus, despite the co-IP data indicating destabilized interactions amongst the MRN subunits in lysates from mre11-W248R cells, our in vivo functional assays indicate that the MRN complex is sufficiently stable to recruit Tel1 to DSBs.Figure 6.


Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair.

Limbo O, Moiani D, Kertokalio A, Wyman C, Tainer JA, Russell P - Nucleic Acids Res. (2012)

Mre11-W248R is proficient in Tel1 activation but partially defective for Ctp1 recruitment. (A) Immunoblot of phosphorylated histone H2A (γH2A) and total H2A with or without treatment with 90 Gy of IR. Graph represents γH2A expression relative to total H2A with error bars representing standard deviation from the mean of three independent experiments. (B) γH2A levels induced specifically by IR treatment determined by subtracting basal levels of γH2A from IR-treated shown in Panel A. (C) Southern blot probing for telomere-associated sequences (TAS1) from genomic DNA isolated from indicated genetic backgrounds and digested with EcoRI (top panel). Ethidium bromide (EtBr)-stained gel shows DNA loading (bottom panel). (D) Chromatin immunoprecipitation of Ctp1-TAP to an HO-induced DNA double-strand break in mre11+ and mre11-W248R backgrounds. Schematic of chromosome I containing HO break site and the relative distances and expected DNA sizes assayed by multiplex PCR is shown on top.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3526295&req=5

gks954-F6: Mre11-W248R is proficient in Tel1 activation but partially defective for Ctp1 recruitment. (A) Immunoblot of phosphorylated histone H2A (γH2A) and total H2A with or without treatment with 90 Gy of IR. Graph represents γH2A expression relative to total H2A with error bars representing standard deviation from the mean of three independent experiments. (B) γH2A levels induced specifically by IR treatment determined by subtracting basal levels of γH2A from IR-treated shown in Panel A. (C) Southern blot probing for telomere-associated sequences (TAS1) from genomic DNA isolated from indicated genetic backgrounds and digested with EcoRI (top panel). Ethidium bromide (EtBr)-stained gel shows DNA loading (bottom panel). (D) Chromatin immunoprecipitation of Ctp1-TAP to an HO-induced DNA double-strand break in mre11+ and mre11-W248R backgrounds. Schematic of chromosome I containing HO break site and the relative distances and expected DNA sizes assayed by multiplex PCR is shown on top.
Mentions: Along with its functions in DNA repair, the MRN complex also recruits the checkpoint kinase ATMTel1 to DNA lesions through interactions with the C-terminus of Nbs1 (3,54,55). ATMTel1 activity therefore depends on the assembly of MRN complex and its capacity to bind DSBs. Thus, phosphorylation of ATMTel1 substrates provides an in vivo readout for these events. The best characterized substrate of Tel1 in fission yeast is histone H2A (analogous to H2AX in mammals), which is also phosphorylated by Rad3ATR (56,57). Therefore, Tel1 activity can be specifically assayed by performing experiments in a rad3Δ background. As expected, IR-induced formation of phospho-H2A (γH2A) was partially maintained in rad3Δ and tel1Δ single mutants but was abolished in the rad3Δ tel1Δ double mutant (Figure 6A and B). γH2A was absent in mre11Δ rad3Δ cells, showing that Mre11 is required for Tel1 activity at DSBs (3). Importantly, γH2A was formed in mre11-W248R rad3Δ cells (Figure 6A and B). Thus, despite the co-IP data indicating destabilized interactions amongst the MRN subunits in lysates from mre11-W248R cells, our in vivo functional assays indicate that the MRN complex is sufficiently stable to recruit Tel1 to DSBs.Figure 6.

Bottom Line: X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions.The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure.Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA.

ABSTRACT
The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.

Show MeSH
Related in: MedlinePlus