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Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados G, Blanco L, Samper E, Bernad A - PLoS Genet. (2009)

Bottom Line: In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development.Hematopoietic progenitors were reduced both in number and in expansion potential.Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

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DSB repair is impaired in Polμ−/− mice.A. Numbers of γ-H2AX foci staining per nuclei in WT (solid bar) or Polμ−/− (open bar) BM and spleen cells. B. Representative confocal images showing γ-H2AX foci (Red, Cy3) in WT and Polμ−/− BM cells (blue staining, DAPI). C. Dot plots showing the comet length of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells. The comet assay was performed in neutral conditions (1×TBE) to assess the relative levels of DNA double strand breakage (DSB). D. Representative fluorescence images of comet assay gels from C. E. Reduced proliferation capacity and premature senescence of cultured Polμ−/− MEF. The 3T3 growth assays show the cumulative increase cell number versus passage in primary (passage 3 at the start of the experiment) WT (closed squares) and Polμ−/− (open triangles) cells. Note that WT MEF enter senescence around division 10, but Polμ−/− MEF stop proliferating by division 4. F. Representative image of a Polμ−/− MEF metaphase spread stained by TEL-FISH. The telomeres are stained with a FITC-labeled (Green) PNA probe and chromosomes are counterstained with DAPI (blue). Asterisks indicate specific chromosomal defects: br (break), r (radial configuration). All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g004: DSB repair is impaired in Polμ−/− mice.A. Numbers of γ-H2AX foci staining per nuclei in WT (solid bar) or Polμ−/− (open bar) BM and spleen cells. B. Representative confocal images showing γ-H2AX foci (Red, Cy3) in WT and Polμ−/− BM cells (blue staining, DAPI). C. Dot plots showing the comet length of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells. The comet assay was performed in neutral conditions (1×TBE) to assess the relative levels of DNA double strand breakage (DSB). D. Representative fluorescence images of comet assay gels from C. E. Reduced proliferation capacity and premature senescence of cultured Polμ−/− MEF. The 3T3 growth assays show the cumulative increase cell number versus passage in primary (passage 3 at the start of the experiment) WT (closed squares) and Polμ−/− (open triangles) cells. Note that WT MEF enter senescence around division 10, but Polμ−/− MEF stop proliferating by division 4. F. Representative image of a Polμ−/− MEF metaphase spread stained by TEL-FISH. The telomeres are stained with a FITC-labeled (Green) PNA probe and chromosomes are counterstained with DAPI (blue). Asterisks indicate specific chromosomal defects: br (break), r (radial configuration). All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: One possible cause of reduced Polμ−/− hematopoietic precursors numbers is impaired DSB repair. To test this, we stained nucleated cells from bone marrow and spleen for phosphorylated γ-H2AX, a marker of DNA double strand breaks [34]. Compared with wt, Polμ−/− bone marrow had a significantly higher number of DSB per nucleus (3.8 fold, p<0.05; Figure 4 A,B); in Polμ−/− spleen cells (mostly non-proliferating) a smaller increase was observed that did not reach significance (Figure 4A). This suggests that Polμ is required for DSB repair in cycling cells. We confirmed the higher DSB incidence in Polμ−/− bone marrow cells by comet assay, which revealed significantly longer comet tails in Polμ−/− BM cells (p<0.001; Figure 4C,D).


Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados G, Blanco L, Samper E, Bernad A - PLoS Genet. (2009)

DSB repair is impaired in Polμ−/− mice.A. Numbers of γ-H2AX foci staining per nuclei in WT (solid bar) or Polμ−/− (open bar) BM and spleen cells. B. Representative confocal images showing γ-H2AX foci (Red, Cy3) in WT and Polμ−/− BM cells (blue staining, DAPI). C. Dot plots showing the comet length of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells. The comet assay was performed in neutral conditions (1×TBE) to assess the relative levels of DNA double strand breakage (DSB). D. Representative fluorescence images of comet assay gels from C. E. Reduced proliferation capacity and premature senescence of cultured Polμ−/− MEF. The 3T3 growth assays show the cumulative increase cell number versus passage in primary (passage 3 at the start of the experiment) WT (closed squares) and Polμ−/− (open triangles) cells. Note that WT MEF enter senescence around division 10, but Polμ−/− MEF stop proliferating by division 4. F. Representative image of a Polμ−/− MEF metaphase spread stained by TEL-FISH. The telomeres are stained with a FITC-labeled (Green) PNA probe and chromosomes are counterstained with DAPI (blue). Asterisks indicate specific chromosomal defects: br (break), r (radial configuration). All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g004: DSB repair is impaired in Polμ−/− mice.A. Numbers of γ-H2AX foci staining per nuclei in WT (solid bar) or Polμ−/− (open bar) BM and spleen cells. B. Representative confocal images showing γ-H2AX foci (Red, Cy3) in WT and Polμ−/− BM cells (blue staining, DAPI). C. Dot plots showing the comet length of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells. The comet assay was performed in neutral conditions (1×TBE) to assess the relative levels of DNA double strand breakage (DSB). D. Representative fluorescence images of comet assay gels from C. E. Reduced proliferation capacity and premature senescence of cultured Polμ−/− MEF. The 3T3 growth assays show the cumulative increase cell number versus passage in primary (passage 3 at the start of the experiment) WT (closed squares) and Polμ−/− (open triangles) cells. Note that WT MEF enter senescence around division 10, but Polμ−/− MEF stop proliferating by division 4. F. Representative image of a Polμ−/− MEF metaphase spread stained by TEL-FISH. The telomeres are stained with a FITC-labeled (Green) PNA probe and chromosomes are counterstained with DAPI (blue). Asterisks indicate specific chromosomal defects: br (break), r (radial configuration). All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: One possible cause of reduced Polμ−/− hematopoietic precursors numbers is impaired DSB repair. To test this, we stained nucleated cells from bone marrow and spleen for phosphorylated γ-H2AX, a marker of DNA double strand breaks [34]. Compared with wt, Polμ−/− bone marrow had a significantly higher number of DSB per nucleus (3.8 fold, p<0.05; Figure 4 A,B); in Polμ−/− spleen cells (mostly non-proliferating) a smaller increase was observed that did not reach significance (Figure 4A). This suggests that Polμ is required for DSB repair in cycling cells. We confirmed the higher DSB incidence in Polμ−/− bone marrow cells by comet assay, which revealed significantly longer comet tails in Polμ−/− BM cells (p<0.001; Figure 4C,D).

Bottom Line: In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development.Hematopoietic progenitors were reduced both in number and in expansion potential.Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

Show MeSH
Related in: MedlinePlus