Limits...
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.

Show MeSH
Mre11 depletion does not affect NHEJ-mediated generation of multimers. (A) Southern blot analysis of NHEJ products generated by incubation of blunt end + 5′-PSS substrate in the indicated depleted egg cytosol extracts at a substrate input concentration of 0.1 (left), 1 (middle), or 10 (right) ng/μl. Data in the left panel are derived from two segments of the same gel (as indicated by the dividing line). (B) Blunt end + 5′-PSS substrate was incubated at 1 ng/μl in untreated egg cytosol that had been supplemented with the recombinant MRN complex, and NHEJ products were analyzed by Southern blot hybridization. M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle; OC, open circle.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171289&req=5

fig3: Mre11 depletion does not affect NHEJ-mediated generation of multimers. (A) Southern blot analysis of NHEJ products generated by incubation of blunt end + 5′-PSS substrate in the indicated depleted egg cytosol extracts at a substrate input concentration of 0.1 (left), 1 (middle), or 10 (right) ng/μl. Data in the left panel are derived from two segments of the same gel (as indicated by the dividing line). (B) Blunt end + 5′-PSS substrate was incubated at 1 ng/μl in untreated egg cytosol that had been supplemented with the recombinant MRN complex, and NHEJ products were analyzed by Southern blot hybridization. M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle; OC, open circle.

Mentions: Next, we determined whether the formation of intermolecular products (dimers and multimers) that were preferentially observed after Ku70 depletion required Mre11. To that end, we depleted Ku70 and Mre11 simultaneously (Fig. 1 C). The formation of intermolecular NHEJ products (LD and M) in Ku70-depleted extracts (Fig. 3 A, lane 8) was not inhibited after further Mre11 depletion (Fig. 3 A, lane 12). Together, these data show that Mre11 is not required for the formation of intramolecular (CC) or intermolecular products (LD and M).


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

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

Mre11 depletion does not affect NHEJ-mediated generation of multimers. (A) Southern blot analysis of NHEJ products generated by incubation of blunt end + 5′-PSS substrate in the indicated depleted egg cytosol extracts at a substrate input concentration of 0.1 (left), 1 (middle), or 10 (right) ng/μl. Data in the left panel are derived from two segments of the same gel (as indicated by the dividing line). (B) Blunt end + 5′-PSS substrate was incubated at 1 ng/μl in untreated egg cytosol that had been supplemented with the recombinant MRN complex, and NHEJ products were analyzed by Southern blot hybridization. M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle; OC, open circle.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171289&req=5

fig3: Mre11 depletion does not affect NHEJ-mediated generation of multimers. (A) Southern blot analysis of NHEJ products generated by incubation of blunt end + 5′-PSS substrate in the indicated depleted egg cytosol extracts at a substrate input concentration of 0.1 (left), 1 (middle), or 10 (right) ng/μl. Data in the left panel are derived from two segments of the same gel (as indicated by the dividing line). (B) Blunt end + 5′-PSS substrate was incubated at 1 ng/μl in untreated egg cytosol that had been supplemented with the recombinant MRN complex, and NHEJ products were analyzed by Southern blot hybridization. M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle; OC, open circle.
Mentions: Next, we determined whether the formation of intermolecular products (dimers and multimers) that were preferentially observed after Ku70 depletion required Mre11. To that end, we depleted Ku70 and Mre11 simultaneously (Fig. 1 C). The formation of intermolecular NHEJ products (LD and M) in Ku70-depleted extracts (Fig. 3 A, lane 8) was not inhibited after further Mre11 depletion (Fig. 3 A, lane 12). Together, these data show that Mre11 is not required for the formation of intramolecular (CC) or intermolecular products (LD and M).

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