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Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11.

Di Virgilio M, Gautier J - J. Cell Biol. (2005)

Bottom Line: In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities.Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products.Finally, Ku70-independent end-joining events are not affected by Mre11 loss.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University, New York, NY 10032, USA.

ABSTRACT
Mre11-Rad50-Nbs1 (MRN) complex involvement in nonhomologous end joining (NHEJ) is controversial. The MRN complex is required for NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe. In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities. In this study, we analyze the effects of Mre11 loss on the mechanism of vertebrate NHEJ by using a chromatinized plasmid double-strand break (DSB) repair assay in cell-free extracts from Xenopus laevis. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs regardless of the end structure. Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system.

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Mre11 is not required for efficient NHEJ. (A) Representative SYBR gold–stained gel characterization of NHEJ products for substrate types nonmatching 3′-PSS (lanes 5–15) and blunt end + 3′-PSS (lanes 16–26). Lanes 5 and 16 show 10 ng of input SacI-KpnI and SacI-SmaI substrates, respectively. The input in lane 2 corresponds to 10 ng of undigested pBS KS II. mt, mitochondrial DNA from X. laevis extract; M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle. (B) The intensity of the bands corresponding to NHEJ products were quantified using the FluorImager system for each NHEJ reaction in Ku70- and Mre11-depleted extracts and were expressed as percent repair efficiency (see Results). The labeling on the x axis refers to the NHEJ substrate type as indicated in Table SI (available at http://www.jcb.org/cgi/content/full/jcb.200506029/DC1).
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fig2: Mre11 is not required for efficient NHEJ. (A) Representative SYBR gold–stained gel characterization of NHEJ products for substrate types nonmatching 3′-PSS (lanes 5–15) and blunt end + 3′-PSS (lanes 16–26). Lanes 5 and 16 show 10 ng of input SacI-KpnI and SacI-SmaI substrates, respectively. The input in lane 2 corresponds to 10 ng of undigested pBS KS II. mt, mitochondrial DNA from X. laevis extract; M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle. (B) The intensity of the bands corresponding to NHEJ products were quantified using the FluorImager system for each NHEJ reaction in Ku70- and Mre11-depleted extracts and were expressed as percent repair efficiency (see Results). The labeling on the x axis refers to the NHEJ substrate type as indicated in Table SI (available at http://www.jcb.org/cgi/content/full/jcb.200506029/DC1).

Mentions: We investigated the consequences of Mre11 inactivation on NHEJ by depleting Mre11 protein from cell-free extracts with a polyclonal serum specific for X. laevis Mre11 protein (anti-XMre11). We previously used these antibodies to demonstrate that genomic DNA accumulates DSBs during DNA replication in Mre11-depleted extracts (Costanzo et al., 2001). Moreover, Mre11 depletion dramatically impairs the ATM (Axatia telangiectasia mutated)-dependent response to DSBs (Costanzo et al., 2004). Importantly, we show that the former defect is rescued by the Mre11 hypomorphic ATLD3/4 allele, whereas ATM activation is not (Costanzo et al., 2004). This clearly demonstrates that Mre11-associated activity remains in ATLD cells. We used two types of cell-free extracts for our study and established that egg cytosol and membrane-free egg cytosol supported NHEJ with similar efficiency (see Figs. 2 and 4).


Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11.

Di Virgilio M, Gautier J - J. Cell Biol. (2005)

Mre11 is not required for efficient NHEJ. (A) Representative SYBR gold–stained gel characterization of NHEJ products for substrate types nonmatching 3′-PSS (lanes 5–15) and blunt end + 3′-PSS (lanes 16–26). Lanes 5 and 16 show 10 ng of input SacI-KpnI and SacI-SmaI substrates, respectively. The input in lane 2 corresponds to 10 ng of undigested pBS KS II. mt, mitochondrial DNA from X. laevis extract; M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle. (B) The intensity of the bands corresponding to NHEJ products were quantified using the FluorImager system for each NHEJ reaction in Ku70- and Mre11-depleted extracts and were expressed as percent repair efficiency (see Results). The labeling on the x axis refers to the NHEJ substrate type as indicated in Table SI (available at http://www.jcb.org/cgi/content/full/jcb.200506029/DC1).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171289&req=5

fig2: Mre11 is not required for efficient NHEJ. (A) Representative SYBR gold–stained gel characterization of NHEJ products for substrate types nonmatching 3′-PSS (lanes 5–15) and blunt end + 3′-PSS (lanes 16–26). Lanes 5 and 16 show 10 ng of input SacI-KpnI and SacI-SmaI substrates, respectively. The input in lane 2 corresponds to 10 ng of undigested pBS KS II. mt, mitochondrial DNA from X. laevis extract; M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle. (B) The intensity of the bands corresponding to NHEJ products were quantified using the FluorImager system for each NHEJ reaction in Ku70- and Mre11-depleted extracts and were expressed as percent repair efficiency (see Results). The labeling on the x axis refers to the NHEJ substrate type as indicated in Table SI (available at http://www.jcb.org/cgi/content/full/jcb.200506029/DC1).
Mentions: We investigated the consequences of Mre11 inactivation on NHEJ by depleting Mre11 protein from cell-free extracts with a polyclonal serum specific for X. laevis Mre11 protein (anti-XMre11). We previously used these antibodies to demonstrate that genomic DNA accumulates DSBs during DNA replication in Mre11-depleted extracts (Costanzo et al., 2001). Moreover, Mre11 depletion dramatically impairs the ATM (Axatia telangiectasia mutated)-dependent response to DSBs (Costanzo et al., 2004). Importantly, we show that the former defect is rescued by the Mre11 hypomorphic ATLD3/4 allele, whereas ATM activation is not (Costanzo et al., 2004). This clearly demonstrates that Mre11-associated activity remains in ATLD cells. We used two types of cell-free extracts for our study and established that egg cytosol and membrane-free egg cytosol supported NHEJ with similar efficiency (see Figs. 2 and 4).

Bottom Line: In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities.Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products.Finally, Ku70-independent end-joining events are not affected by Mre11 loss.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University, New York, NY 10032, USA.

ABSTRACT
Mre11-Rad50-Nbs1 (MRN) complex involvement in nonhomologous end joining (NHEJ) is controversial. The MRN complex is required for NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe. In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities. In this study, we analyze the effects of Mre11 loss on the mechanism of vertebrate NHEJ by using a chromatinized plasmid double-strand break (DSB) repair assay in cell-free extracts from Xenopus laevis. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs regardless of the end structure. Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system.

Show MeSH