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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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Association of MMR proteins at centromere-associated and Y chromosome repeats is dependent on MutSβ (MSH2–MSH3). (A-C) chromosome localization of MSH2 (red, TRITC) on wild-type SCs, as marked by SYCP3 (green, FITC) in association with the centromere, as marked by CREST labeling (blue, CY5), and the XY bivalent (C, arrow). MSH2 localization occurs at centromeres and the Y chromosome in spermatocytes from wild-type males (A–C; same cell in each panel), and is also found at occasional sites along the chromosome and throughout the chromatin area (see arrowheads in A). (D–F) MSH3 localization follows a similar pattern to MSH2, as shown in D (MSH3 is red, TRITC) and localizes most strongly with the CREST-positive regions (blue, CY5) in E and F, and also localizes to the XY (indicated by the arrow in F). (G–I) Immunogold electron micrographs showing the centromere regions of chromosomes from Msh3−/− (G), Msh4−/− (H), and Msh5−/− (I) spermatocytes in pachynema with MLH3 (12 nm gold beads, green), MSH2 (6 nm gold beads, red), and CREST (18 nm gold beads, blue) showing that the localization of MSH2 and MLH3 to this region is dependent on MSH3, but not on MSH4 and MSH5. (J) Quantitation of EM-gold localization of MLH3 (striped bars), MLH1 (filled bars), and CREST (empty bars) on spermatocyte centromeres from wild-type males, and males that are homozygous for mutations in Msh2, Msh3, Msh5, and Msh6. MLH3 localization is significantly decreased compared with wild type on centromeres from Msh2−/− and Msh3−/− spermatocytes, but not on centromeres from Msh5−/− or Msh6−/− spermatocytes. χ2 analysis of MLH1 and MLH3 localization at spermatocyte centromeres reveals statistically significant differences between wild-type and both Msh2−/− and Msh3−/− males (***P < 0.0002), but not between wild-type and Msh5−/− males (not significant, n.s., P = 0.81). MLH3 levels were slightly increased at the centromeres of Msh6−/− spermatocytes (*P = 0.043).
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fig6: Association of MMR proteins at centromere-associated and Y chromosome repeats is dependent on MutSβ (MSH2–MSH3). (A-C) chromosome localization of MSH2 (red, TRITC) on wild-type SCs, as marked by SYCP3 (green, FITC) in association with the centromere, as marked by CREST labeling (blue, CY5), and the XY bivalent (C, arrow). MSH2 localization occurs at centromeres and the Y chromosome in spermatocytes from wild-type males (A–C; same cell in each panel), and is also found at occasional sites along the chromosome and throughout the chromatin area (see arrowheads in A). (D–F) MSH3 localization follows a similar pattern to MSH2, as shown in D (MSH3 is red, TRITC) and localizes most strongly with the CREST-positive regions (blue, CY5) in E and F, and also localizes to the XY (indicated by the arrow in F). (G–I) Immunogold electron micrographs showing the centromere regions of chromosomes from Msh3−/− (G), Msh4−/− (H), and Msh5−/− (I) spermatocytes in pachynema with MLH3 (12 nm gold beads, green), MSH2 (6 nm gold beads, red), and CREST (18 nm gold beads, blue) showing that the localization of MSH2 and MLH3 to this region is dependent on MSH3, but not on MSH4 and MSH5. (J) Quantitation of EM-gold localization of MLH3 (striped bars), MLH1 (filled bars), and CREST (empty bars) on spermatocyte centromeres from wild-type males, and males that are homozygous for mutations in Msh2, Msh3, Msh5, and Msh6. MLH3 localization is significantly decreased compared with wild type on centromeres from Msh2−/− and Msh3−/− spermatocytes, but not on centromeres from Msh5−/− or Msh6−/− spermatocytes. χ2 analysis of MLH1 and MLH3 localization at spermatocyte centromeres reveals statistically significant differences between wild-type and both Msh2−/− and Msh3−/− males (***P < 0.0002), but not between wild-type and Msh5−/− males (not significant, n.s., P = 0.81). MLH3 levels were slightly increased at the centromeres of Msh6−/− spermatocytes (*P = 0.043).

Mentions: Immunofluorescence analysis of chromosome preparations from wild-type spermatocytes demonstrated that MSH2 and MSH3 localize strongly to the centromere (Fig. 6, A–F) and on the sex chromosomes (Fig. 6, C and F, arrows), and that this localization is as frequent in wild-type spermatocytes as it is in Pms2−/− males (Fig. 7, A–C and J). These observations indicate that the association of MSH2–MSH3-containing complexes appear to be a feature of normal prophase I and are not specifically up-regulated in the absence of PMS2. No association of MSH2 with these genomic regions is observed in spermatocytes from Msh3−/− males either by immunofluorescence or EM (Fig. 6 G and Fig. 7, D–F), indicating that MSH3 is required for MSH2 localization to these sites. By contrast, immunofluorescence and immunogold localization of MSH2 at the centromere is not affected in spermatocytes from Msh4−/−, Msh5−/−, or Msh6−/− males (Fig. 6, H and I, and Fig. 7, G–J). MSH2, and to a lesser degree MSH3, localizes along the chromosomes, but at an intensity that is far lower than that seen at the centromere (Fig. 6, A and D, arrowheads; Fig. 7, A and G, arrowheads). This interstitial localization could be the result of canonical MMR activity.


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)

Association of MMR proteins at centromere-associated and Y chromosome repeats is dependent on MutSβ (MSH2–MSH3). (A-C) chromosome localization of MSH2 (red, TRITC) on wild-type SCs, as marked by SYCP3 (green, FITC) in association with the centromere, as marked by CREST labeling (blue, CY5), and the XY bivalent (C, arrow). MSH2 localization occurs at centromeres and the Y chromosome in spermatocytes from wild-type males (A–C; same cell in each panel), and is also found at occasional sites along the chromosome and throughout the chromatin area (see arrowheads in A). (D–F) MSH3 localization follows a similar pattern to MSH2, as shown in D (MSH3 is red, TRITC) and localizes most strongly with the CREST-positive regions (blue, CY5) in E and F, and also localizes to the XY (indicated by the arrow in F). (G–I) Immunogold electron micrographs showing the centromere regions of chromosomes from Msh3−/− (G), Msh4−/− (H), and Msh5−/− (I) spermatocytes in pachynema with MLH3 (12 nm gold beads, green), MSH2 (6 nm gold beads, red), and CREST (18 nm gold beads, blue) showing that the localization of MSH2 and MLH3 to this region is dependent on MSH3, but not on MSH4 and MSH5. (J) Quantitation of EM-gold localization of MLH3 (striped bars), MLH1 (filled bars), and CREST (empty bars) on spermatocyte centromeres from wild-type males, and males that are homozygous for mutations in Msh2, Msh3, Msh5, and Msh6. MLH3 localization is significantly decreased compared with wild type on centromeres from Msh2−/− and Msh3−/− spermatocytes, but not on centromeres from Msh5−/− or Msh6−/− spermatocytes. χ2 analysis of MLH1 and MLH3 localization at spermatocyte centromeres reveals statistically significant differences between wild-type and both Msh2−/− and Msh3−/− males (***P < 0.0002), but not between wild-type and Msh5−/− males (not significant, n.s., P = 0.81). MLH3 levels were slightly increased at the centromeres of Msh6−/− spermatocytes (*P = 0.043).
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fig6: Association of MMR proteins at centromere-associated and Y chromosome repeats is dependent on MutSβ (MSH2–MSH3). (A-C) chromosome localization of MSH2 (red, TRITC) on wild-type SCs, as marked by SYCP3 (green, FITC) in association with the centromere, as marked by CREST labeling (blue, CY5), and the XY bivalent (C, arrow). MSH2 localization occurs at centromeres and the Y chromosome in spermatocytes from wild-type males (A–C; same cell in each panel), and is also found at occasional sites along the chromosome and throughout the chromatin area (see arrowheads in A). (D–F) MSH3 localization follows a similar pattern to MSH2, as shown in D (MSH3 is red, TRITC) and localizes most strongly with the CREST-positive regions (blue, CY5) in E and F, and also localizes to the XY (indicated by the arrow in F). (G–I) Immunogold electron micrographs showing the centromere regions of chromosomes from Msh3−/− (G), Msh4−/− (H), and Msh5−/− (I) spermatocytes in pachynema with MLH3 (12 nm gold beads, green), MSH2 (6 nm gold beads, red), and CREST (18 nm gold beads, blue) showing that the localization of MSH2 and MLH3 to this region is dependent on MSH3, but not on MSH4 and MSH5. (J) Quantitation of EM-gold localization of MLH3 (striped bars), MLH1 (filled bars), and CREST (empty bars) on spermatocyte centromeres from wild-type males, and males that are homozygous for mutations in Msh2, Msh3, Msh5, and Msh6. MLH3 localization is significantly decreased compared with wild type on centromeres from Msh2−/− and Msh3−/− spermatocytes, but not on centromeres from Msh5−/− or Msh6−/− spermatocytes. χ2 analysis of MLH1 and MLH3 localization at spermatocyte centromeres reveals statistically significant differences between wild-type and both Msh2−/− and Msh3−/− males (***P < 0.0002), but not between wild-type and Msh5−/− males (not significant, n.s., P = 0.81). MLH3 levels were slightly increased at the centromeres of Msh6−/− spermatocytes (*P = 0.043).
Mentions: Immunofluorescence analysis of chromosome preparations from wild-type spermatocytes demonstrated that MSH2 and MSH3 localize strongly to the centromere (Fig. 6, A–F) and on the sex chromosomes (Fig. 6, C and F, arrows), and that this localization is as frequent in wild-type spermatocytes as it is in Pms2−/− males (Fig. 7, A–C and J). These observations indicate that the association of MSH2–MSH3-containing complexes appear to be a feature of normal prophase I and are not specifically up-regulated in the absence of PMS2. No association of MSH2 with these genomic regions is observed in spermatocytes from Msh3−/− males either by immunofluorescence or EM (Fig. 6 G and Fig. 7, D–F), indicating that MSH3 is required for MSH2 localization to these sites. By contrast, immunofluorescence and immunogold localization of MSH2 at the centromere is not affected in spermatocytes from Msh4−/−, Msh5−/−, or Msh6−/− males (Fig. 6, H and I, and Fig. 7, G–J). MSH2, and to a lesser degree MSH3, localizes along the chromosomes, but at an intensity that is far lower than that seen at the centromere (Fig. 6, A and D, arrowheads; Fig. 7, A and G, arrowheads). This interstitial localization could be the result of canonical MMR activity.

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