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Multiple functions of MRN in end-joining pathways during isotype class switching.

Dinkelmann M, Spehalski E, Stoneham T, Buis J, Wu Y, Sekiguchi JM, Ferguson DO - Nat. Struct. Mol. Biol. (2009)

Bottom Line: The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood.MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways.We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.

ABSTRACT
The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

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Mre11 deficiencies in the B lymphocyte lineage(a) Four germline mouse alleles of Mre11: Mre11 wild type (Mre11+), Mre11 conditional (Mre11cond), Mre11  (Mre11−), and Mre11 deficient in nuclease activities (Mre11H129N). Mre11 exon 5 (gray rectangle), intronic sequence (black line), LoxP sites (triangles), histidine to asparagine mutation at amino acid 129 (asterisk). Base pair (bp) numbers indicate allele specific PCR products resulting from the two primers depicted (arrows)9. (b) Location of the invariant active site histidine within nuclease motif III. Mouse histidine 129 was changed to asparagine as described9. (c) PCR analyses distinguishing the four Mre11 germline alleles. Conversion of Mre11cond to Mre11− is detected only in sites containing substantial numbers of B lymphocytes (BM - bone marrow, LN - lymph node, SP - whole spleen, BC - enriched B cell population from spleen), but not in kidney (KD). Primers used depicted in (a). (d) Western blot analyses of MRN components and AID in splenic B lymphocytes from mice harboring one allele of CD19-Cre. GAPDH was used as a protein loading control. Left - resting B cells, right - B cells stimulated to undergo class switch recombination for 4 days in culture. Each mouse contains the Mre11 allele indicated, and a second allele which is Mre11− in the B lineage (Mre11cond elsewhere).
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Figure 1: Mre11 deficiencies in the B lymphocyte lineage(a) Four germline mouse alleles of Mre11: Mre11 wild type (Mre11+), Mre11 conditional (Mre11cond), Mre11 (Mre11−), and Mre11 deficient in nuclease activities (Mre11H129N). Mre11 exon 5 (gray rectangle), intronic sequence (black line), LoxP sites (triangles), histidine to asparagine mutation at amino acid 129 (asterisk). Base pair (bp) numbers indicate allele specific PCR products resulting from the two primers depicted (arrows)9. (b) Location of the invariant active site histidine within nuclease motif III. Mouse histidine 129 was changed to asparagine as described9. (c) PCR analyses distinguishing the four Mre11 germline alleles. Conversion of Mre11cond to Mre11− is detected only in sites containing substantial numbers of B lymphocytes (BM - bone marrow, LN - lymph node, SP - whole spleen, BC - enriched B cell population from spleen), but not in kidney (KD). Primers used depicted in (a). (d) Western blot analyses of MRN components and AID in splenic B lymphocytes from mice harboring one allele of CD19-Cre. GAPDH was used as a protein loading control. Left - resting B cells, right - B cells stimulated to undergo class switch recombination for 4 days in culture. Each mouse contains the Mre11 allele indicated, and a second allele which is Mre11− in the B lineage (Mre11cond elsewhere).

Mentions: We have utilized an engineered mouse allele of Mre11 that functions as wild type until conditionally inactivated via the Cre recombinase (Fig. 1a). Previous work has shown that conversion of conditional (Mre11cond/−) to (Mre11−/−) not only causes depletion of Mre11, but of the entire MRN complex, likely due to instability of the other components9. In addition, we have utilized an allele containing a targeted single amino acid change (Mre11H129N) that eliminates the endo- and exonuclease activities of Mre11 without disrupting the MRN complex, or its ability to sense DSBs and activate ATM (Fig. 1a, b)9. Studies of the Mre11H129N allele revealed that the nuclease activities of Mre11 are required for DSB repair via the HR pathway9.


Multiple functions of MRN in end-joining pathways during isotype class switching.

Dinkelmann M, Spehalski E, Stoneham T, Buis J, Wu Y, Sekiguchi JM, Ferguson DO - Nat. Struct. Mol. Biol. (2009)

Mre11 deficiencies in the B lymphocyte lineage(a) Four germline mouse alleles of Mre11: Mre11 wild type (Mre11+), Mre11 conditional (Mre11cond), Mre11  (Mre11−), and Mre11 deficient in nuclease activities (Mre11H129N). Mre11 exon 5 (gray rectangle), intronic sequence (black line), LoxP sites (triangles), histidine to asparagine mutation at amino acid 129 (asterisk). Base pair (bp) numbers indicate allele specific PCR products resulting from the two primers depicted (arrows)9. (b) Location of the invariant active site histidine within nuclease motif III. Mouse histidine 129 was changed to asparagine as described9. (c) PCR analyses distinguishing the four Mre11 germline alleles. Conversion of Mre11cond to Mre11− is detected only in sites containing substantial numbers of B lymphocytes (BM - bone marrow, LN - lymph node, SP - whole spleen, BC - enriched B cell population from spleen), but not in kidney (KD). Primers used depicted in (a). (d) Western blot analyses of MRN components and AID in splenic B lymphocytes from mice harboring one allele of CD19-Cre. GAPDH was used as a protein loading control. Left - resting B cells, right - B cells stimulated to undergo class switch recombination for 4 days in culture. Each mouse contains the Mre11 allele indicated, and a second allele which is Mre11− in the B lineage (Mre11cond elsewhere).
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Related In: Results  -  Collection

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Figure 1: Mre11 deficiencies in the B lymphocyte lineage(a) Four germline mouse alleles of Mre11: Mre11 wild type (Mre11+), Mre11 conditional (Mre11cond), Mre11 (Mre11−), and Mre11 deficient in nuclease activities (Mre11H129N). Mre11 exon 5 (gray rectangle), intronic sequence (black line), LoxP sites (triangles), histidine to asparagine mutation at amino acid 129 (asterisk). Base pair (bp) numbers indicate allele specific PCR products resulting from the two primers depicted (arrows)9. (b) Location of the invariant active site histidine within nuclease motif III. Mouse histidine 129 was changed to asparagine as described9. (c) PCR analyses distinguishing the four Mre11 germline alleles. Conversion of Mre11cond to Mre11− is detected only in sites containing substantial numbers of B lymphocytes (BM - bone marrow, LN - lymph node, SP - whole spleen, BC - enriched B cell population from spleen), but not in kidney (KD). Primers used depicted in (a). (d) Western blot analyses of MRN components and AID in splenic B lymphocytes from mice harboring one allele of CD19-Cre. GAPDH was used as a protein loading control. Left - resting B cells, right - B cells stimulated to undergo class switch recombination for 4 days in culture. Each mouse contains the Mre11 allele indicated, and a second allele which is Mre11− in the B lineage (Mre11cond elsewhere).
Mentions: We have utilized an engineered mouse allele of Mre11 that functions as wild type until conditionally inactivated via the Cre recombinase (Fig. 1a). Previous work has shown that conversion of conditional (Mre11cond/−) to (Mre11−/−) not only causes depletion of Mre11, but of the entire MRN complex, likely due to instability of the other components9. In addition, we have utilized an allele containing a targeted single amino acid change (Mre11H129N) that eliminates the endo- and exonuclease activities of Mre11 without disrupting the MRN complex, or its ability to sense DSBs and activate ATM (Fig. 1a, b)9. Studies of the Mre11H129N allele revealed that the nuclease activities of Mre11 are required for DSB repair via the HR pathway9.

Bottom Line: The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood.MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways.We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.

ABSTRACT
The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

Show MeSH
Related in: MedlinePlus