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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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Localization of MMR complexes on the mouse Y chromosome during prophase I. (A) Immunogold-EM micrograph of an XY bivalent from a Pms2−/− spermatocyte showing the PAR, the entire Y chromosome and part of the X chromosome. MLH3 localization is marked by the presence of 12 nm gold beads, showing a large dense focus on the Y chromosome. The centromere (cen) is marked with CREST labeled with 18 nm gold beads, and the SC is counterstained with phosphotungstic acid. (B) Representative slot blot analysis of Y chromosome repeat sequences immunoprecipitated with antibodies against MutSβ (combined MSH2–MSH3 antibodies; Oncogene Research Products), MLH3, and rabbit IgG (Mock control). (C) Quantitation of ChIP experiment in B, expressed as a percentage of input DNA.
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fig5: Localization of MMR complexes on the mouse Y chromosome during prophase I. (A) Immunogold-EM micrograph of an XY bivalent from a Pms2−/− spermatocyte showing the PAR, the entire Y chromosome and part of the X chromosome. MLH3 localization is marked by the presence of 12 nm gold beads, showing a large dense focus on the Y chromosome. The centromere (cen) is marked with CREST labeled with 18 nm gold beads, and the SC is counterstained with phosphotungstic acid. (B) Representative slot blot analysis of Y chromosome repeat sequences immunoprecipitated with antibodies against MutSβ (combined MSH2–MSH3 antibodies; Oncogene Research Products), MLH3, and rabbit IgG (Mock control). (C) Quantitation of ChIP experiment in B, expressed as a percentage of input DNA.

Mentions: To investigate the association of the MMR machinery with other repetitive sequences in the genome, we explored the localization of MLH3 across the Y chromosome, which in humans and mice contains large palindromic repeat sequences (Bishop et al., 1985; Singh et al., 1994; Rozen et al., 2003; Skaletsky et al., 2003) and which, like the centromere, is refractory to reciprocal recombination except at the X-Y pseudoautosomal region (PAR). MLH3 localizes in dense foci along the Y chromosome in some pachytene spermatocytes from wild-type, Pms2−/− and Mlh1−/− males (Fig. 5 A, Pms2−/− example shown), but the localization is not consistent among spermatocyte populations. In total, of the more than 500 nuclei examined from Pms2−/− males, ∼10% exhibited the Y chromosome accumulation of MLH3. No MLH1 association was found along the Y chromosome in wild-type or in Pms2−/− spermatocytes (unpublished data). ChIP analysis confirmed the localization of MLH3 (Fig. 5, B and C), and the absence of MLH1 (unpublished data), on Y chromosome sequences from Pms2−/− males.


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)

Localization of MMR complexes on the mouse Y chromosome during prophase I. (A) Immunogold-EM micrograph of an XY bivalent from a Pms2−/− spermatocyte showing the PAR, the entire Y chromosome and part of the X chromosome. MLH3 localization is marked by the presence of 12 nm gold beads, showing a large dense focus on the Y chromosome. The centromere (cen) is marked with CREST labeled with 18 nm gold beads, and the SC is counterstained with phosphotungstic acid. (B) Representative slot blot analysis of Y chromosome repeat sequences immunoprecipitated with antibodies against MutSβ (combined MSH2–MSH3 antibodies; Oncogene Research Products), MLH3, and rabbit IgG (Mock control). (C) Quantitation of ChIP experiment in B, expressed as a percentage of input DNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171243&req=5

fig5: Localization of MMR complexes on the mouse Y chromosome during prophase I. (A) Immunogold-EM micrograph of an XY bivalent from a Pms2−/− spermatocyte showing the PAR, the entire Y chromosome and part of the X chromosome. MLH3 localization is marked by the presence of 12 nm gold beads, showing a large dense focus on the Y chromosome. The centromere (cen) is marked with CREST labeled with 18 nm gold beads, and the SC is counterstained with phosphotungstic acid. (B) Representative slot blot analysis of Y chromosome repeat sequences immunoprecipitated with antibodies against MutSβ (combined MSH2–MSH3 antibodies; Oncogene Research Products), MLH3, and rabbit IgG (Mock control). (C) Quantitation of ChIP experiment in B, expressed as a percentage of input DNA.
Mentions: To investigate the association of the MMR machinery with other repetitive sequences in the genome, we explored the localization of MLH3 across the Y chromosome, which in humans and mice contains large palindromic repeat sequences (Bishop et al., 1985; Singh et al., 1994; Rozen et al., 2003; Skaletsky et al., 2003) and which, like the centromere, is refractory to reciprocal recombination except at the X-Y pseudoautosomal region (PAR). MLH3 localizes in dense foci along the Y chromosome in some pachytene spermatocytes from wild-type, Pms2−/− and Mlh1−/− males (Fig. 5 A, Pms2−/− example shown), but the localization is not consistent among spermatocyte populations. In total, of the more than 500 nuclei examined from Pms2−/− males, ∼10% exhibited the Y chromosome accumulation of MLH3. No MLH1 association was found along the Y chromosome in wild-type or in Pms2−/− spermatocytes (unpublished data). ChIP analysis confirmed the localization of MLH3 (Fig. 5, B and C), and the absence of MLH1 (unpublished data), on Y chromosome sequences from Pms2−/− males.

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