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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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Expression pattern of MLH3 and MLH1 in spermatocytes from wild-type mice and those with targeted mutations of the MutL homologues. (A) MLH1 (top; green, FITC) localizes to spermatocyte SCs (SYCP3: red, TRITC) from mid to late pachynema but is not present in early diplonema. MLH3 localizes to SCs earlier than MLH1, during early pachynema, and persists until late pachynema. Centromeres are labeled with CREST (blue, CY5). (B) Quantitation of MLH1 (black bars) and MLH3 (striped bars) on wild-type spermatocyte SCs confirms the earlier localization of MLH3. (C) Immunogold-EM localization of MLH3 (6 nm gold grains) and MLH1 (12 nm gold grains) on Pms2−/− spermatocytes demonstrates colocalization to electron-dense MNs (inset) during mid-pachynema. Centromeres (cen) are labeled with CREST (18 nm gold grains). Bar, 200 nm. (D) Quantitation of MLH1 (black bars) and MLH3 (striped bars) localization indicates that MN frequency, as measured by MLH1/3 labeling, is unaffected, or slightly elevated, in Pms2−/− spermatocytes. (E) Quantitation of MLH1 and MLH3 foci in spermatocytes from mice with targeted deletions of Mlh1, Mlh3, and Pms2 indicates that even though MLH3 can bind to SCs in the absence of MLH1, MLH1 is incapable of localizing in the absence of MLH3. Neither MLH1 nor MLH3 localization is reduced in the absence of PMS2. (F) Immunogold-EM showing MLH3 (12 nm gold grains) localization to MNs (inset) in the absence of MLH1. Centromeres (cen) are localized with CREST (18 nm gold grains). Bar, 500 nm. Error bars indicate SD.
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fig1: Expression pattern of MLH3 and MLH1 in spermatocytes from wild-type mice and those with targeted mutations of the MutL homologues. (A) MLH1 (top; green, FITC) localizes to spermatocyte SCs (SYCP3: red, TRITC) from mid to late pachynema but is not present in early diplonema. MLH3 localizes to SCs earlier than MLH1, during early pachynema, and persists until late pachynema. Centromeres are labeled with CREST (blue, CY5). (B) Quantitation of MLH1 (black bars) and MLH3 (striped bars) on wild-type spermatocyte SCs confirms the earlier localization of MLH3. (C) Immunogold-EM localization of MLH3 (6 nm gold grains) and MLH1 (12 nm gold grains) on Pms2−/− spermatocytes demonstrates colocalization to electron-dense MNs (inset) during mid-pachynema. Centromeres (cen) are labeled with CREST (18 nm gold grains). Bar, 200 nm. (D) Quantitation of MLH1 (black bars) and MLH3 (striped bars) localization indicates that MN frequency, as measured by MLH1/3 labeling, is unaffected, or slightly elevated, in Pms2−/− spermatocytes. (E) Quantitation of MLH1 and MLH3 foci in spermatocytes from mice with targeted deletions of Mlh1, Mlh3, and Pms2 indicates that even though MLH3 can bind to SCs in the absence of MLH1, MLH1 is incapable of localizing in the absence of MLH3. Neither MLH1 nor MLH3 localization is reduced in the absence of PMS2. (F) Immunogold-EM showing MLH3 (12 nm gold grains) localization to MNs (inset) in the absence of MLH1. Centromeres (cen) are localized with CREST (18 nm gold grains). Bar, 500 nm. Error bars indicate SD.

Mentions: The synaptonemal complex (SC) is the tripartite protein structure that forms a continuous filament structure along homologous chromosomes and tethers them together during prophase I (Page and Hawley, 2004). The SC also serves as the docking site for key recombinational proteins of the MN. To assess the dynamics of MutL homologue accumulation through prophase I, MLH1 and MLH3 were localized to SCs of mouse spermatocytes. MLH3 foci are found on the SC of wild-type spermatocytes from early pachynema (Fig. 1, A and B), but MLH1 does not associate with MLH3 until mid-pachynema (Fig. 1, A and B). The early MLH3 foci probably account for the few instances of MLH3-positive, MLH1-negative foci reported in our previous studies (Lipkin et al., 2002), that we suggest then recruit MLH1 in mid-pachynema. MLH1 and MLH3 persist through to late pachynema and are rarely seen at diplonema. The number of MLH1–MLH3 foci correlates well with the predicted number of crossovers in mouse spermatocytes (Anderson et al., 1999), suggesting that MLH1–MLH3 marks most, if not all, of the reciprocal recombination sites during mouse meiosis, and making it less likely that MLH1–PMS2 heterodimers are marking another subset of such sites.


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)

Expression pattern of MLH3 and MLH1 in spermatocytes from wild-type mice and those with targeted mutations of the MutL homologues. (A) MLH1 (top; green, FITC) localizes to spermatocyte SCs (SYCP3: red, TRITC) from mid to late pachynema but is not present in early diplonema. MLH3 localizes to SCs earlier than MLH1, during early pachynema, and persists until late pachynema. Centromeres are labeled with CREST (blue, CY5). (B) Quantitation of MLH1 (black bars) and MLH3 (striped bars) on wild-type spermatocyte SCs confirms the earlier localization of MLH3. (C) Immunogold-EM localization of MLH3 (6 nm gold grains) and MLH1 (12 nm gold grains) on Pms2−/− spermatocytes demonstrates colocalization to electron-dense MNs (inset) during mid-pachynema. Centromeres (cen) are labeled with CREST (18 nm gold grains). Bar, 200 nm. (D) Quantitation of MLH1 (black bars) and MLH3 (striped bars) localization indicates that MN frequency, as measured by MLH1/3 labeling, is unaffected, or slightly elevated, in Pms2−/− spermatocytes. (E) Quantitation of MLH1 and MLH3 foci in spermatocytes from mice with targeted deletions of Mlh1, Mlh3, and Pms2 indicates that even though MLH3 can bind to SCs in the absence of MLH1, MLH1 is incapable of localizing in the absence of MLH3. Neither MLH1 nor MLH3 localization is reduced in the absence of PMS2. (F) Immunogold-EM showing MLH3 (12 nm gold grains) localization to MNs (inset) in the absence of MLH1. Centromeres (cen) are localized with CREST (18 nm gold grains). Bar, 500 nm. Error bars indicate SD.
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Related In: Results  -  Collection

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fig1: Expression pattern of MLH3 and MLH1 in spermatocytes from wild-type mice and those with targeted mutations of the MutL homologues. (A) MLH1 (top; green, FITC) localizes to spermatocyte SCs (SYCP3: red, TRITC) from mid to late pachynema but is not present in early diplonema. MLH3 localizes to SCs earlier than MLH1, during early pachynema, and persists until late pachynema. Centromeres are labeled with CREST (blue, CY5). (B) Quantitation of MLH1 (black bars) and MLH3 (striped bars) on wild-type spermatocyte SCs confirms the earlier localization of MLH3. (C) Immunogold-EM localization of MLH3 (6 nm gold grains) and MLH1 (12 nm gold grains) on Pms2−/− spermatocytes demonstrates colocalization to electron-dense MNs (inset) during mid-pachynema. Centromeres (cen) are labeled with CREST (18 nm gold grains). Bar, 200 nm. (D) Quantitation of MLH1 (black bars) and MLH3 (striped bars) localization indicates that MN frequency, as measured by MLH1/3 labeling, is unaffected, or slightly elevated, in Pms2−/− spermatocytes. (E) Quantitation of MLH1 and MLH3 foci in spermatocytes from mice with targeted deletions of Mlh1, Mlh3, and Pms2 indicates that even though MLH3 can bind to SCs in the absence of MLH1, MLH1 is incapable of localizing in the absence of MLH3. Neither MLH1 nor MLH3 localization is reduced in the absence of PMS2. (F) Immunogold-EM showing MLH3 (12 nm gold grains) localization to MNs (inset) in the absence of MLH1. Centromeres (cen) are localized with CREST (18 nm gold grains). Bar, 500 nm. Error bars indicate SD.
Mentions: The synaptonemal complex (SC) is the tripartite protein structure that forms a continuous filament structure along homologous chromosomes and tethers them together during prophase I (Page and Hawley, 2004). The SC also serves as the docking site for key recombinational proteins of the MN. To assess the dynamics of MutL homologue accumulation through prophase I, MLH1 and MLH3 were localized to SCs of mouse spermatocytes. MLH3 foci are found on the SC of wild-type spermatocytes from early pachynema (Fig. 1, A and B), but MLH1 does not associate with MLH3 until mid-pachynema (Fig. 1, A and B). The early MLH3 foci probably account for the few instances of MLH3-positive, MLH1-negative foci reported in our previous studies (Lipkin et al., 2002), that we suggest then recruit MLH1 in mid-pachynema. MLH1 and MLH3 persist through to late pachynema and are rarely seen at diplonema. The number of MLH1–MLH3 foci correlates well with the predicted number of crossovers in mouse spermatocytes (Anderson et al., 1999), suggesting that MLH1–MLH3 marks most, if not all, of the reciprocal recombination sites during mouse meiosis, and making it less likely that MLH1–PMS2 heterodimers are marking another subset of such sites.

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