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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

MCM8-9 are required for the localization of MRN complex on HR repair sites.(a,b) MCM8-9 and MRE11 required for forming RPA foci in cisplatin-treated cells. The western blot (a) and quantification of RPA70 foci-positive cells (b) after knockdown of indicated proteins in U2OS cells. ***P<0.005; Student's t-test. (c) MRN complex coimmunoprecipitates with MCM8-9. Endogenous MCM9 (left) or Rad50 (right) was immunoprecipitated (IP) from HEK293T cells using indicated antibodies in the presence of EtBr and immunoblotted for indicated proteins. (d) Co-localization of Flag-MCM8 and MRE11 in nuclear foci after exposure to cisplatin. Cells were pre-extracted for immunostaining. Scale bar, 10 μm. (e) Defect of MRN recruitment to I-SceI cut site in MCM8- or MCM9-depleted cells. ChIP assays were performed using indicated antibodies in HeLa DR13-9 cells 18 h after transfecting plasmid expressing I-SceI. Fold signal at cut site relative to site 2 kb upstream as described in Fig. 1c. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (f) Decrease of MRN foci-positive cells in MCM8- or MCM9-depleted cells. Representative images (left) and % of MRN foci-positive cells (right). Cells having over 20 foci >0.5 μm diameter were counted as positive. Scale bar, 10 μm. ***P<0.005, **P<0.01; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole; EV, empty vector; IgG, immunoglobulin G.
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f3: MCM8-9 are required for the localization of MRN complex on HR repair sites.(a,b) MCM8-9 and MRE11 required for forming RPA foci in cisplatin-treated cells. The western blot (a) and quantification of RPA70 foci-positive cells (b) after knockdown of indicated proteins in U2OS cells. ***P<0.005; Student's t-test. (c) MRN complex coimmunoprecipitates with MCM8-9. Endogenous MCM9 (left) or Rad50 (right) was immunoprecipitated (IP) from HEK293T cells using indicated antibodies in the presence of EtBr and immunoblotted for indicated proteins. (d) Co-localization of Flag-MCM8 and MRE11 in nuclear foci after exposure to cisplatin. Cells were pre-extracted for immunostaining. Scale bar, 10 μm. (e) Defect of MRN recruitment to I-SceI cut site in MCM8- or MCM9-depleted cells. ChIP assays were performed using indicated antibodies in HeLa DR13-9 cells 18 h after transfecting plasmid expressing I-SceI. Fold signal at cut site relative to site 2 kb upstream as described in Fig. 1c. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (f) Decrease of MRN foci-positive cells in MCM8- or MCM9-depleted cells. Representative images (left) and % of MRN foci-positive cells (right). Cells having over 20 foci >0.5 μm diameter were counted as positive. Scale bar, 10 μm. ***P<0.005, **P<0.01; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole; EV, empty vector; IgG, immunoglobulin G.

Mentions: To screen for the nuclease(s) requiring MCM8-9 activity for ssDNA formation, we first tested which nucleases were required for RPA focus formation after cisplatin treatment. The knockdown of MRE11, but not EXO1 and/or DNA2, suppressed cisplatin-induced RPA foci formation to the same extent as seen after MCM8-9 depletion (Fig. 3a,b and Supplementary Fig. 3a,b). This result suggested that MCM8-9 might work during the initial DNA resection alongside the MRN complex. Therefore, we examined whether MRN and MCM8-9 physically associate with each other. Indeed, MCM9 immunoprecipitates contained not only MCM8 but also MRE11, NBS1 and RAD50 (Fig. 3c, left), and conversely RAD50 immunoprecipitates contained not only its cofactors, NBS1 and MRE11, but also MCM8-9 (Fig. 3c, right). The co-immunoprecipitation was performed in the presence of the DNA intercalating chemical, ethidium bromide (EtBr), suggesting that the MCM8-9 and MRN did not interact via a bridging DNA molecule. Furthermore, MRE11 foci generated by cisplatin treatment co-localized with Flag-MCM8 foci (Fig. 3d). Chromatin immunoprecipitation (ChIP) assays on HeLa DR13-9 cells showed that all of the MRN components normally accumulated on the I-SceI cut site following the expression of I-SceI, but this association was impaired on depletion of MCM8 or MCM9 (Fig. 3e). In a parallel experiment, siMCM8 or siMCM9 decreased the number of cisplatin-induced foci formed by all MRN complex members (Fig. 3f). On the other hand, the recruitment of CtIP to cisplatin induced foci and to I-SceI cut sites was not affected by depletion of MCM8-9 (Supplementary Fig. 4a,b). Therefore, in multiple experimental systems, MCM8-9 is required for the proper localization of the MRN complex to DSB sites for DNA resection.


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)

MCM8-9 are required for the localization of MRN complex on HR repair sites.(a,b) MCM8-9 and MRE11 required for forming RPA foci in cisplatin-treated cells. The western blot (a) and quantification of RPA70 foci-positive cells (b) after knockdown of indicated proteins in U2OS cells. ***P<0.005; Student's t-test. (c) MRN complex coimmunoprecipitates with MCM8-9. Endogenous MCM9 (left) or Rad50 (right) was immunoprecipitated (IP) from HEK293T cells using indicated antibodies in the presence of EtBr and immunoblotted for indicated proteins. (d) Co-localization of Flag-MCM8 and MRE11 in nuclear foci after exposure to cisplatin. Cells were pre-extracted for immunostaining. Scale bar, 10 μm. (e) Defect of MRN recruitment to I-SceI cut site in MCM8- or MCM9-depleted cells. ChIP assays were performed using indicated antibodies in HeLa DR13-9 cells 18 h after transfecting plasmid expressing I-SceI. Fold signal at cut site relative to site 2 kb upstream as described in Fig. 1c. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (f) Decrease of MRN foci-positive cells in MCM8- or MCM9-depleted cells. Representative images (left) and % of MRN foci-positive cells (right). Cells having over 20 foci >0.5 μm diameter were counted as positive. Scale bar, 10 μm. ***P<0.005, **P<0.01; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole; EV, empty vector; IgG, immunoglobulin G.
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f3: MCM8-9 are required for the localization of MRN complex on HR repair sites.(a,b) MCM8-9 and MRE11 required for forming RPA foci in cisplatin-treated cells. The western blot (a) and quantification of RPA70 foci-positive cells (b) after knockdown of indicated proteins in U2OS cells. ***P<0.005; Student's t-test. (c) MRN complex coimmunoprecipitates with MCM8-9. Endogenous MCM9 (left) or Rad50 (right) was immunoprecipitated (IP) from HEK293T cells using indicated antibodies in the presence of EtBr and immunoblotted for indicated proteins. (d) Co-localization of Flag-MCM8 and MRE11 in nuclear foci after exposure to cisplatin. Cells were pre-extracted for immunostaining. Scale bar, 10 μm. (e) Defect of MRN recruitment to I-SceI cut site in MCM8- or MCM9-depleted cells. ChIP assays were performed using indicated antibodies in HeLa DR13-9 cells 18 h after transfecting plasmid expressing I-SceI. Fold signal at cut site relative to site 2 kb upstream as described in Fig. 1c. ***P<0.005, **P<0.01, *P<0.05; Student's t-test. (f) Decrease of MRN foci-positive cells in MCM8- or MCM9-depleted cells. Representative images (left) and % of MRN foci-positive cells (right). Cells having over 20 foci >0.5 μm diameter were counted as positive. Scale bar, 10 μm. ***P<0.005, **P<0.01; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole; EV, empty vector; IgG, immunoglobulin G.
Mentions: To screen for the nuclease(s) requiring MCM8-9 activity for ssDNA formation, we first tested which nucleases were required for RPA focus formation after cisplatin treatment. The knockdown of MRE11, but not EXO1 and/or DNA2, suppressed cisplatin-induced RPA foci formation to the same extent as seen after MCM8-9 depletion (Fig. 3a,b and Supplementary Fig. 3a,b). This result suggested that MCM8-9 might work during the initial DNA resection alongside the MRN complex. Therefore, we examined whether MRN and MCM8-9 physically associate with each other. Indeed, MCM9 immunoprecipitates contained not only MCM8 but also MRE11, NBS1 and RAD50 (Fig. 3c, left), and conversely RAD50 immunoprecipitates contained not only its cofactors, NBS1 and MRE11, but also MCM8-9 (Fig. 3c, right). The co-immunoprecipitation was performed in the presence of the DNA intercalating chemical, ethidium bromide (EtBr), suggesting that the MCM8-9 and MRN did not interact via a bridging DNA molecule. Furthermore, MRE11 foci generated by cisplatin treatment co-localized with Flag-MCM8 foci (Fig. 3d). Chromatin immunoprecipitation (ChIP) assays on HeLa DR13-9 cells showed that all of the MRN components normally accumulated on the I-SceI cut site following the expression of I-SceI, but this association was impaired on depletion of MCM8 or MCM9 (Fig. 3e). In a parallel experiment, siMCM8 or siMCM9 decreased the number of cisplatin-induced foci formed by all MRN complex members (Fig. 3f). On the other hand, the recruitment of CtIP to cisplatin induced foci and to I-SceI cut sites was not affected by depletion of MCM8-9 (Supplementary Fig. 4a,b). Therefore, in multiple experimental systems, MCM8-9 is required for the proper localization of the MRN complex to DSB sites for DNA resection.

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