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Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.

Kolas NK, Svetlanov A, Lenzi ML, Macaluso FP, Lipkin SM, Liskay RM, Greally J, Edelmann W, Cohen PE - J. Cell Biol. (2005)

Bottom Line: Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects.This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice.The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

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MLH3 immunolocalizes to a proportion of wild-type and Mlh1−/− spermatocyte centromeres, and both MLH3 and MLH1 localize to the centromeres of Pms2−/−spermatocytes but not Pms2−/− oocytes. (A–D) Immunogold-EM localization of MLH1 (12 nm gold grains, red arrows), MLH3 (6 nm gold grains, green arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosomes of Pms2−/− (A), wild-type (B), Mlh1−/− (C), and Mlh3−/− (D) spermatocytes. Bars: (A, B, and D) 250 nm; (C) C 500 nm. All SCs are counterstained with phosphotungstic acid. (E) Quantitation of MMR protein localization to centromeres from Mlh1+/+, Mlh1−/−, Mlh3+/+, Mlh3−/−, Pms2+/+, and Pms2−/− spermatocytes. Counts were obtained from immunogold-EM micrographs exemplified in A–D and represent those centromeres showing more than four gold grains for each antibody staining. At least 100 centromeres were counted for each genotype from three mice. Percentages represent pooled counts from all these mice. MLH3 (striped bars) immunolocalizes to 15–25% of wild-type and Mlh1−/−, but not Mlh3−/−, spermatocyte centromeres. MLH1 (black bars) does not localize in any appreciable amount to centromeres in wild-type, Mlh1−/− or Mlh3−/− spermatocytes. CREST (white bars) localizes to 100% of the centromeres. Pms2−/− spermatocyte centromeres show a statistically significant increase in localization of both MLH1 and MLH3 in comparison with the Pms2+/+ and all other mouse strains observed. (F) Quantitation of distal (noncentromeric) telomere localization (more than four gold grains) indicates that the localization of MutL homologues is specific to the centromere. (G) EM micrograph of MLH1 (6 nm gold grains) and MLH3 (12 nm gold grains) on SCs from Pms2−/− oocytes indicates that, whereas MLH3 and MLH1 localize normally to MNs (bottom inset), these MutL homologues fail to localize to the centromere (cen), labeled with CREST (18 nm gold grains; top inset). Bar, 200 nm.
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fig2: MLH3 immunolocalizes to a proportion of wild-type and Mlh1−/− spermatocyte centromeres, and both MLH3 and MLH1 localize to the centromeres of Pms2−/−spermatocytes but not Pms2−/− oocytes. (A–D) Immunogold-EM localization of MLH1 (12 nm gold grains, red arrows), MLH3 (6 nm gold grains, green arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosomes of Pms2−/− (A), wild-type (B), Mlh1−/− (C), and Mlh3−/− (D) spermatocytes. Bars: (A, B, and D) 250 nm; (C) C 500 nm. All SCs are counterstained with phosphotungstic acid. (E) Quantitation of MMR protein localization to centromeres from Mlh1+/+, Mlh1−/−, Mlh3+/+, Mlh3−/−, Pms2+/+, and Pms2−/− spermatocytes. Counts were obtained from immunogold-EM micrographs exemplified in A–D and represent those centromeres showing more than four gold grains for each antibody staining. At least 100 centromeres were counted for each genotype from three mice. Percentages represent pooled counts from all these mice. MLH3 (striped bars) immunolocalizes to 15–25% of wild-type and Mlh1−/−, but not Mlh3−/−, spermatocyte centromeres. MLH1 (black bars) does not localize in any appreciable amount to centromeres in wild-type, Mlh1−/− or Mlh3−/− spermatocytes. CREST (white bars) localizes to 100% of the centromeres. Pms2−/− spermatocyte centromeres show a statistically significant increase in localization of both MLH1 and MLH3 in comparison with the Pms2+/+ and all other mouse strains observed. (F) Quantitation of distal (noncentromeric) telomere localization (more than four gold grains) indicates that the localization of MutL homologues is specific to the centromere. (G) EM micrograph of MLH1 (6 nm gold grains) and MLH3 (12 nm gold grains) on SCs from Pms2−/− oocytes indicates that, whereas MLH3 and MLH1 localize normally to MNs (bottom inset), these MutL homologues fail to localize to the centromere (cen), labeled with CREST (18 nm gold grains; top inset). Bar, 200 nm.

Mentions: In view of the apparently normal recombination frequency in spermatocytes of Pms2−/− males (Fig. 1; Qin et al., 2002), we investigated whether other MMR-related functions might be disrupted in these animals. Examination of the SCs from Pms2−/− males by EM revealed an accumulation of both MLH1 and MLH3 close to the centromere (Fig. 2 A), a region that is usually not subject to crossing over (Broman et al., 1998; Froenicke et al., 2002) and is therefore not expected to be positive for this MutL complex. Examination of the centromere regions from other MMR-deficient mouse strains revealed similar, but reduced frequency of, localization of MLH3 in spermatocytes from wild-type (Fig. 2 B) and Mlh1−/− males (Fig. 2 C), but not from Mlh3−/− males (Fig. 2 D). In wild-type and Mlh1−/− males, MLH3 associated with ∼15–25% of all spermatocyte autosomal centromeres. Little if any MLH1 was associated with centromere regions in wild-type, Mlh1−/−, or Mlh3−/− mice (Fig. 2, B–E). The localization of MLH3 at centromeres increased to include >85% of centromere regions in spermatocytes from Pms2−/− males, and colocalized at most of these sites (>65% of total centromeres) with MLH1 (Fig. 2 E). The EM images of immunogold-labeled spermatocyte spread preparations were supported by similar localization at the immunofluorescence level (Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200506170/DC1), but was much weaker using fluorescently-labeled secondary antibodies (which are monoclonal antibodies) than using gold-labeled secondaries (polyclonal antibodies). The EM analysis involves a higher concentration of primary antibody, coupled with DNaseI digestion of heterochromatin to improve accessibility of the antibodies to the centromere sites, perhaps explaining the difference in staining intensity.


Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.

Kolas NK, Svetlanov A, Lenzi ML, Macaluso FP, Lipkin SM, Liskay RM, Greally J, Edelmann W, Cohen PE - J. Cell Biol. (2005)

MLH3 immunolocalizes to a proportion of wild-type and Mlh1−/− spermatocyte centromeres, and both MLH3 and MLH1 localize to the centromeres of Pms2−/−spermatocytes but not Pms2−/− oocytes. (A–D) Immunogold-EM localization of MLH1 (12 nm gold grains, red arrows), MLH3 (6 nm gold grains, green arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosomes of Pms2−/− (A), wild-type (B), Mlh1−/− (C), and Mlh3−/− (D) spermatocytes. Bars: (A, B, and D) 250 nm; (C) C 500 nm. All SCs are counterstained with phosphotungstic acid. (E) Quantitation of MMR protein localization to centromeres from Mlh1+/+, Mlh1−/−, Mlh3+/+, Mlh3−/−, Pms2+/+, and Pms2−/− spermatocytes. Counts were obtained from immunogold-EM micrographs exemplified in A–D and represent those centromeres showing more than four gold grains for each antibody staining. At least 100 centromeres were counted for each genotype from three mice. Percentages represent pooled counts from all these mice. MLH3 (striped bars) immunolocalizes to 15–25% of wild-type and Mlh1−/−, but not Mlh3−/−, spermatocyte centromeres. MLH1 (black bars) does not localize in any appreciable amount to centromeres in wild-type, Mlh1−/− or Mlh3−/− spermatocytes. CREST (white bars) localizes to 100% of the centromeres. Pms2−/− spermatocyte centromeres show a statistically significant increase in localization of both MLH1 and MLH3 in comparison with the Pms2+/+ and all other mouse strains observed. (F) Quantitation of distal (noncentromeric) telomere localization (more than four gold grains) indicates that the localization of MutL homologues is specific to the centromere. (G) EM micrograph of MLH1 (6 nm gold grains) and MLH3 (12 nm gold grains) on SCs from Pms2−/− oocytes indicates that, whereas MLH3 and MLH1 localize normally to MNs (bottom inset), these MutL homologues fail to localize to the centromere (cen), labeled with CREST (18 nm gold grains; top inset). Bar, 200 nm.
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fig2: MLH3 immunolocalizes to a proportion of wild-type and Mlh1−/− spermatocyte centromeres, and both MLH3 and MLH1 localize to the centromeres of Pms2−/−spermatocytes but not Pms2−/− oocytes. (A–D) Immunogold-EM localization of MLH1 (12 nm gold grains, red arrows), MLH3 (6 nm gold grains, green arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosomes of Pms2−/− (A), wild-type (B), Mlh1−/− (C), and Mlh3−/− (D) spermatocytes. Bars: (A, B, and D) 250 nm; (C) C 500 nm. All SCs are counterstained with phosphotungstic acid. (E) Quantitation of MMR protein localization to centromeres from Mlh1+/+, Mlh1−/−, Mlh3+/+, Mlh3−/−, Pms2+/+, and Pms2−/− spermatocytes. Counts were obtained from immunogold-EM micrographs exemplified in A–D and represent those centromeres showing more than four gold grains for each antibody staining. At least 100 centromeres were counted for each genotype from three mice. Percentages represent pooled counts from all these mice. MLH3 (striped bars) immunolocalizes to 15–25% of wild-type and Mlh1−/−, but not Mlh3−/−, spermatocyte centromeres. MLH1 (black bars) does not localize in any appreciable amount to centromeres in wild-type, Mlh1−/− or Mlh3−/− spermatocytes. CREST (white bars) localizes to 100% of the centromeres. Pms2−/− spermatocyte centromeres show a statistically significant increase in localization of both MLH1 and MLH3 in comparison with the Pms2+/+ and all other mouse strains observed. (F) Quantitation of distal (noncentromeric) telomere localization (more than four gold grains) indicates that the localization of MutL homologues is specific to the centromere. (G) EM micrograph of MLH1 (6 nm gold grains) and MLH3 (12 nm gold grains) on SCs from Pms2−/− oocytes indicates that, whereas MLH3 and MLH1 localize normally to MNs (bottom inset), these MutL homologues fail to localize to the centromere (cen), labeled with CREST (18 nm gold grains; top inset). Bar, 200 nm.
Mentions: In view of the apparently normal recombination frequency in spermatocytes of Pms2−/− males (Fig. 1; Qin et al., 2002), we investigated whether other MMR-related functions might be disrupted in these animals. Examination of the SCs from Pms2−/− males by EM revealed an accumulation of both MLH1 and MLH3 close to the centromere (Fig. 2 A), a region that is usually not subject to crossing over (Broman et al., 1998; Froenicke et al., 2002) and is therefore not expected to be positive for this MutL complex. Examination of the centromere regions from other MMR-deficient mouse strains revealed similar, but reduced frequency of, localization of MLH3 in spermatocytes from wild-type (Fig. 2 B) and Mlh1−/− males (Fig. 2 C), but not from Mlh3−/− males (Fig. 2 D). In wild-type and Mlh1−/− males, MLH3 associated with ∼15–25% of all spermatocyte autosomal centromeres. Little if any MLH1 was associated with centromere regions in wild-type, Mlh1−/−, or Mlh3−/− mice (Fig. 2, B–E). The localization of MLH3 at centromeres increased to include >85% of centromere regions in spermatocytes from Pms2−/− males, and colocalized at most of these sites (>65% of total centromeres) with MLH1 (Fig. 2 E). The EM images of immunogold-labeled spermatocyte spread preparations were supported by similar localization at the immunofluorescence level (Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200506170/DC1), but was much weaker using fluorescently-labeled secondary antibodies (which are monoclonal antibodies) than using gold-labeled secondaries (polyclonal antibodies). The EM analysis involves a higher concentration of primary antibody, coupled with DNaseI digestion of heterochromatin to improve accessibility of the antibodies to the centromere sites, perhaps explaining the difference in staining intensity.

Bottom Line: Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects.This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice.The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

Show MeSH
Related in: MedlinePlus