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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 activity of MCM9 is essential for the function of MRN nuclease.(a) Decrease of MRN endonuclease activity in MCM8-9-depleted cells. In vitro endonuclease assay using MRN purified by anti-FLAG from U2OS cells stably expressing FLAG-NBS1. ØX174 ssDNA substrate was incubated for indicated times. Top: EtBr stain of reaction products shows the substrate C, circular ØX174 and the product L, linearized ØX174. Bottom: quantification of linearized ssDNA with ImageJ software, normalized to the level in the 0-min lane. ***P<0.005, *P<0.05; Student's t-test. (b) Silver stain of purified recombinant Xenopus MCM8-9 complex. WT, wild type-MCM8 and -MCM9; WA, WA mutant MCM9. (c) WT/WT MCM8-9 has more nuclease activity than WT/WA MCM8-9. The in vitro nuclease assay was performed using indicated amounts of the recombinant MCM8-9 or bovine serum albumin (BSA). (d) Immunodepletion of MRE11, as detected by immunoblot, from purified recombinant MCM8-9. Immunodepletion was done by incubating anti-human MRE11 antibody with indicated recombinant proteins. B, before immunodepletion; A, after immunodepletion; I, the total immunoprecipitate. (e) Reduced nuclease activity of WT/WT MCM8-9 after immunodepletion of MRE11 (A), compared with that before immunodepletion (B). Amount of full-length linear DNA remaining was quantified after 60 min of an in vitro nuclease assay with 5 nM of BSA or purified MCM8-9 before or after immunodepletion of MRE11. The y axis shows the ratio of the residual substrate relative to that at the 0-min point. ***P<0.005, *P<0.05; Student's t-test. All error bars represent s.d. of the mean from triplicates.
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f5: ATPase activity of MCM9 is essential for the function of MRN nuclease.(a) Decrease of MRN endonuclease activity in MCM8-9-depleted cells. In vitro endonuclease assay using MRN purified by anti-FLAG from U2OS cells stably expressing FLAG-NBS1. ØX174 ssDNA substrate was incubated for indicated times. Top: EtBr stain of reaction products shows the substrate C, circular ØX174 and the product L, linearized ØX174. Bottom: quantification of linearized ssDNA with ImageJ software, normalized to the level in the 0-min lane. ***P<0.005, *P<0.05; Student's t-test. (b) Silver stain of purified recombinant Xenopus MCM8-9 complex. WT, wild type-MCM8 and -MCM9; WA, WA mutant MCM9. (c) WT/WT MCM8-9 has more nuclease activity than WT/WA MCM8-9. The in vitro nuclease assay was performed using indicated amounts of the recombinant MCM8-9 or bovine serum albumin (BSA). (d) Immunodepletion of MRE11, as detected by immunoblot, from purified recombinant MCM8-9. Immunodepletion was done by incubating anti-human MRE11 antibody with indicated recombinant proteins. B, before immunodepletion; A, after immunodepletion; I, the total immunoprecipitate. (e) Reduced nuclease activity of WT/WT MCM8-9 after immunodepletion of MRE11 (A), compared with that before immunodepletion (B). Amount of full-length linear DNA remaining was quantified after 60 min of an in vitro nuclease assay with 5 nM of BSA or purified MCM8-9 before or after immunodepletion of MRE11. The y axis shows the ratio of the residual substrate relative to that at the 0-min point. ***P<0.005, *P<0.05; Student's t-test. All error bars represent s.d. of the mean from triplicates.

Mentions: MRE11 endonuclease initiates resection at DSBs before HR8. We purified the MRN complex from U2OS cells stably expressing FLAG-NBS1 and tested its endonuclease activity on circular ØX174 ssDNA (Fig. 5a and Supplementary Fig. 7a). MCM8 knockdown decreased the endonuclease activity of the immunoprecipitated MRN proteins, suggesting that human MCM8-9 is required for optimal nuclease activity of the MRN complex. Note that the nuclease activity of purified MRN complex was inhibited by MRE11 inhibitor, mirin (Supplementary Fig 7b). Thus, the nuclease activity in the MRN immunoprecipitate was mainly due to MRE11, although we cannot rule out the presence of other contaminating endonucleases.


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 activity of MCM9 is essential for the function of MRN nuclease.(a) Decrease of MRN endonuclease activity in MCM8-9-depleted cells. In vitro endonuclease assay using MRN purified by anti-FLAG from U2OS cells stably expressing FLAG-NBS1. ØX174 ssDNA substrate was incubated for indicated times. Top: EtBr stain of reaction products shows the substrate C, circular ØX174 and the product L, linearized ØX174. Bottom: quantification of linearized ssDNA with ImageJ software, normalized to the level in the 0-min lane. ***P<0.005, *P<0.05; Student's t-test. (b) Silver stain of purified recombinant Xenopus MCM8-9 complex. WT, wild type-MCM8 and -MCM9; WA, WA mutant MCM9. (c) WT/WT MCM8-9 has more nuclease activity than WT/WA MCM8-9. The in vitro nuclease assay was performed using indicated amounts of the recombinant MCM8-9 or bovine serum albumin (BSA). (d) Immunodepletion of MRE11, as detected by immunoblot, from purified recombinant MCM8-9. Immunodepletion was done by incubating anti-human MRE11 antibody with indicated recombinant proteins. B, before immunodepletion; A, after immunodepletion; I, the total immunoprecipitate. (e) Reduced nuclease activity of WT/WT MCM8-9 after immunodepletion of MRE11 (A), compared with that before immunodepletion (B). Amount of full-length linear DNA remaining was quantified after 60 min of an in vitro nuclease assay with 5 nM of BSA or purified MCM8-9 before or after immunodepletion of MRE11. The y axis shows the ratio of the residual substrate relative to that at the 0-min point. ***P<0.005, *P<0.05; Student's t-test. All error bars represent s.d. of the mean from triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525285&req=5

f5: ATPase activity of MCM9 is essential for the function of MRN nuclease.(a) Decrease of MRN endonuclease activity in MCM8-9-depleted cells. In vitro endonuclease assay using MRN purified by anti-FLAG from U2OS cells stably expressing FLAG-NBS1. ØX174 ssDNA substrate was incubated for indicated times. Top: EtBr stain of reaction products shows the substrate C, circular ØX174 and the product L, linearized ØX174. Bottom: quantification of linearized ssDNA with ImageJ software, normalized to the level in the 0-min lane. ***P<0.005, *P<0.05; Student's t-test. (b) Silver stain of purified recombinant Xenopus MCM8-9 complex. WT, wild type-MCM8 and -MCM9; WA, WA mutant MCM9. (c) WT/WT MCM8-9 has more nuclease activity than WT/WA MCM8-9. The in vitro nuclease assay was performed using indicated amounts of the recombinant MCM8-9 or bovine serum albumin (BSA). (d) Immunodepletion of MRE11, as detected by immunoblot, from purified recombinant MCM8-9. Immunodepletion was done by incubating anti-human MRE11 antibody with indicated recombinant proteins. B, before immunodepletion; A, after immunodepletion; I, the total immunoprecipitate. (e) Reduced nuclease activity of WT/WT MCM8-9 after immunodepletion of MRE11 (A), compared with that before immunodepletion (B). Amount of full-length linear DNA remaining was quantified after 60 min of an in vitro nuclease assay with 5 nM of BSA or purified MCM8-9 before or after immunodepletion of MRE11. The y axis shows the ratio of the residual substrate relative to that at the 0-min point. ***P<0.005, *P<0.05; Student's t-test. All error bars represent s.d. of the mean from triplicates.
Mentions: MRE11 endonuclease initiates resection at DSBs before HR8. We purified the MRN complex from U2OS cells stably expressing FLAG-NBS1 and tested its endonuclease activity on circular ØX174 ssDNA (Fig. 5a and Supplementary Fig. 7a). MCM8 knockdown decreased the endonuclease activity of the immunoprecipitated MRN proteins, suggesting that human MCM8-9 is required for optimal nuclease activity of the MRN complex. Note that the nuclease activity of purified MRN complex was inhibited by MRE11 inhibitor, mirin (Supplementary Fig 7b). Thus, the nuclease activity in the MRN immunoprecipitate was mainly due to MRE11, although we cannot rule out the presence of other contaminating endonucleases.

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