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MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.

Lee KY, Im JS, Shibata E, Park J, Handa N, Kowalczykowski SC, Dutta A - Nat Commun (2015)

Bottom Line: MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs).MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs.A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Jordan Hall, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908 USA.

ABSTRACT
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.

No MeSH data available.


Related in: MedlinePlus

ATPase motif of MCM9 is essential for HR repair and the interaction with MRE11 protein.(a,b) Mutation on ATPase motif of MCM8-9 decreases RPA (top) or MRE11 (bottom) foci formation. U2OS cells supported by Walker A- (WA) or Walker B (WB) mutants of MCM8 (a) or MCM9 (b) were treated with cisplatin after knockdown of the endogenous protein, and foci-positive cells were counted as described previously. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (c) WA- or WB mutant of MCM9 cannot restore resistance to cisplatin after knockdown of endogenous MCM9. Cell viability was measured by colony count at day 5 after cisplatin treatment . ***P<0.005; Student's t-test. (d) WA- or WB mutant MCM9 cannot rescue HR. HR assays were performed in HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9. HR efficiency was measured by normalizing the percentage of GFP-positive cells of each sample to that of the siGL2-treated cells. ***P<0.005; Student's t-test. (e) WA- or WB mutant MCM9 cannot recruit MRE11 to I-SceI cut site. ChIP was done using HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9 after knockdown of endogenous MCM9. Signal at cut site expressed relative to -2 kb site. **P<0.01, *P<0.05; Student's t-test. (f) WA- or WB mutant MCM9 does not co-immunoprecipitate MRE11 from HEK293T cells transfected by the indicated plasmids expressing MCM9. (g) Decrease in nuclease associated with WA- or WB mutant MCM9. DNA products visualized after in vitro nuclease assay for 90 min with epitope-tagged MCM9 immunoprecipitated (IP) from cells transfected with indicated plasmids and siRNAs as described in Methods section. Cells were treated with 40 μM cisplatin for 4 h before harvest. All error bars represent s.d. of the mean from triplicates. EV, empty vector; WT, wild type.
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f4: ATPase motif of MCM9 is essential for HR repair and the interaction with MRE11 protein.(a,b) Mutation on ATPase motif of MCM8-9 decreases RPA (top) or MRE11 (bottom) foci formation. U2OS cells supported by Walker A- (WA) or Walker B (WB) mutants of MCM8 (a) or MCM9 (b) were treated with cisplatin after knockdown of the endogenous protein, and foci-positive cells were counted as described previously. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (c) WA- or WB mutant of MCM9 cannot restore resistance to cisplatin after knockdown of endogenous MCM9. Cell viability was measured by colony count at day 5 after cisplatin treatment . ***P<0.005; Student's t-test. (d) WA- or WB mutant MCM9 cannot rescue HR. HR assays were performed in HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9. HR efficiency was measured by normalizing the percentage of GFP-positive cells of each sample to that of the siGL2-treated cells. ***P<0.005; Student's t-test. (e) WA- or WB mutant MCM9 cannot recruit MRE11 to I-SceI cut site. ChIP was done using HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9 after knockdown of endogenous MCM9. Signal at cut site expressed relative to -2 kb site. **P<0.01, *P<0.05; Student's t-test. (f) WA- or WB mutant MCM9 does not co-immunoprecipitate MRE11 from HEK293T cells transfected by the indicated plasmids expressing MCM9. (g) Decrease in nuclease associated with WA- or WB mutant MCM9. DNA products visualized after in vitro nuclease assay for 90 min with epitope-tagged MCM9 immunoprecipitated (IP) from cells transfected with indicated plasmids and siRNAs as described in Methods section. Cells were treated with 40 μM cisplatin for 4 h before harvest. All error bars represent s.d. of the mean from triplicates. EV, empty vector; WT, wild type.

Mentions: MCM8 and MCM9 are related in sequence to all of the components of the MCM2-7 complex, which unwinds duplex DNA during DNA replication23. Human MCM8 and MCM9 contain MCM domains marked by the Walker A (WA) and Walker B (WB) motifs important for ATP binding and hydrolysis. Therefore, we tested whether the ATPase activities of MCM8 or MCM9 were essential for DNA resection in HR repair. U2OS cell lines stably expressing WA or WB motif mutants of siRNA-resistant MCM8 or MCM9 were used. On depletion of endogenous MCM8 or MCM9 by siRNA, WT MCM8 and MCM9 (MCM8r-WT and MCM9r-WT) restored the RPA- and MRE11 foci on cisplatin treatment (Fig. 4a,b). The WA- or WB motif mutants of MCM8 or MCM9 were unable to restore these foci.


MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.

Lee KY, Im JS, Shibata E, Park J, Handa N, Kowalczykowski SC, Dutta A - Nat Commun (2015)

ATPase motif of MCM9 is essential for HR repair and the interaction with MRE11 protein.(a,b) Mutation on ATPase motif of MCM8-9 decreases RPA (top) or MRE11 (bottom) foci formation. U2OS cells supported by Walker A- (WA) or Walker B (WB) mutants of MCM8 (a) or MCM9 (b) were treated with cisplatin after knockdown of the endogenous protein, and foci-positive cells were counted as described previously. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (c) WA- or WB mutant of MCM9 cannot restore resistance to cisplatin after knockdown of endogenous MCM9. Cell viability was measured by colony count at day 5 after cisplatin treatment . ***P<0.005; Student's t-test. (d) WA- or WB mutant MCM9 cannot rescue HR. HR assays were performed in HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9. HR efficiency was measured by normalizing the percentage of GFP-positive cells of each sample to that of the siGL2-treated cells. ***P<0.005; Student's t-test. (e) WA- or WB mutant MCM9 cannot recruit MRE11 to I-SceI cut site. ChIP was done using HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9 after knockdown of endogenous MCM9. Signal at cut site expressed relative to -2 kb site. **P<0.01, *P<0.05; Student's t-test. (f) WA- or WB mutant MCM9 does not co-immunoprecipitate MRE11 from HEK293T cells transfected by the indicated plasmids expressing MCM9. (g) Decrease in nuclease associated with WA- or WB mutant MCM9. DNA products visualized after in vitro nuclease assay for 90 min with epitope-tagged MCM9 immunoprecipitated (IP) from cells transfected with indicated plasmids and siRNAs as described in Methods section. Cells were treated with 40 μM cisplatin for 4 h before harvest. All error bars represent s.d. of the mean from triplicates. EV, empty vector; WT, wild type.
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Related In: Results  -  Collection

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Show All Figures
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f4: ATPase motif of MCM9 is essential for HR repair and the interaction with MRE11 protein.(a,b) Mutation on ATPase motif of MCM8-9 decreases RPA (top) or MRE11 (bottom) foci formation. U2OS cells supported by Walker A- (WA) or Walker B (WB) mutants of MCM8 (a) or MCM9 (b) were treated with cisplatin after knockdown of the endogenous protein, and foci-positive cells were counted as described previously. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (c) WA- or WB mutant of MCM9 cannot restore resistance to cisplatin after knockdown of endogenous MCM9. Cell viability was measured by colony count at day 5 after cisplatin treatment . ***P<0.005; Student's t-test. (d) WA- or WB mutant MCM9 cannot rescue HR. HR assays were performed in HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9. HR efficiency was measured by normalizing the percentage of GFP-positive cells of each sample to that of the siGL2-treated cells. ***P<0.005; Student's t-test. (e) WA- or WB mutant MCM9 cannot recruit MRE11 to I-SceI cut site. ChIP was done using HeLa DR13-9 cells having stable expression of siRNA-resistant MCM9 after knockdown of endogenous MCM9. Signal at cut site expressed relative to -2 kb site. **P<0.01, *P<0.05; Student's t-test. (f) WA- or WB mutant MCM9 does not co-immunoprecipitate MRE11 from HEK293T cells transfected by the indicated plasmids expressing MCM9. (g) Decrease in nuclease associated with WA- or WB mutant MCM9. DNA products visualized after in vitro nuclease assay for 90 min with epitope-tagged MCM9 immunoprecipitated (IP) from cells transfected with indicated plasmids and siRNAs as described in Methods section. Cells were treated with 40 μM cisplatin for 4 h before harvest. All error bars represent s.d. of the mean from triplicates. EV, empty vector; WT, wild type.
Mentions: MCM8 and MCM9 are related in sequence to all of the components of the MCM2-7 complex, which unwinds duplex DNA during DNA replication23. Human MCM8 and MCM9 contain MCM domains marked by the Walker A (WA) and Walker B (WB) motifs important for ATP binding and hydrolysis. Therefore, we tested whether the ATPase activities of MCM8 or MCM9 were essential for DNA resection in HR repair. U2OS cell lines stably expressing WA or WB motif mutants of siRNA-resistant MCM8 or MCM9 were used. On depletion of endogenous MCM8 or MCM9 by siRNA, WT MCM8 and MCM9 (MCM8r-WT and MCM9r-WT) restored the RPA- and MRE11 foci on cisplatin treatment (Fig. 4a,b). The WA- or WB motif mutants of MCM8 or MCM9 were unable to restore these foci.

Bottom Line: MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs).MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs.A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Jordan Hall, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908 USA.

ABSTRACT
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.

No MeSH data available.


Related in: MedlinePlus