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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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Chromatin immunoprecipitation of centromere-associated repeat sequences by antibodies against MMR proteins. (A) Representative slot blot analysis of major and minor satellite repeat sequences (CEN) performed on crude germ cell lysates immunoprecipitated with antibodies MLH3, CREST, or rabbit IgG (Mock control), compared with input DNA. The same slot blot membrane was stripped and reprobed with a B2 SINE probe as a loading control. (B) Quantitation of three centromere ChIP experiments, expressed as a percentage of input DNA + SD. Statistical analysis was performed using unpaired t-tests (***P < 0.0001). (C) ChIP assay followed by PCR using primers against minor and major satellite sequences performed on purified pachytene cells from Pms2−/− testes. Lanes (from left to right): lane 1, molecular weight marker; lanes 2–5, minor satellite PCR; lanes 6–9, major satellite PCR. M, 100 bp marker; Input, input DNA. 10-fold dilutions of ChIP pull-down substrate DNA was used for minor satellite PCR, DNA for major satellite PCR was undiluted.
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fig4: Chromatin immunoprecipitation of centromere-associated repeat sequences by antibodies against MMR proteins. (A) Representative slot blot analysis of major and minor satellite repeat sequences (CEN) performed on crude germ cell lysates immunoprecipitated with antibodies MLH3, CREST, or rabbit IgG (Mock control), compared with input DNA. The same slot blot membrane was stripped and reprobed with a B2 SINE probe as a loading control. (B) Quantitation of three centromere ChIP experiments, expressed as a percentage of input DNA + SD. Statistical analysis was performed using unpaired t-tests (***P < 0.0001). (C) ChIP assay followed by PCR using primers against minor and major satellite sequences performed on purified pachytene cells from Pms2−/− testes. Lanes (from left to right): lane 1, molecular weight marker; lanes 2–5, minor satellite PCR; lanes 6–9, major satellite PCR. M, 100 bp marker; Input, input DNA. 10-fold dilutions of ChIP pull-down substrate DNA was used for minor satellite PCR, DNA for major satellite PCR was undiluted.

Mentions: ChIP of mixed germ cell extracts from Pms2−/− males with the anti-MLH3 antibody enriched centromeric major/minor satellite sequences above control levels at comparable rates to that seen for polyclonal CREST serum, which recognizes a variety of CENPs (Fig. 4, A and B). The enrichment of centromere repeats in Pms2−/− spermatocytes with anti-MLH3 antibody or with CREST was statistically elevated compared with mock controls (unpaired t tests, P < 0.0001). No enrichment of similar regions was found in ChIP extracts from Mlh1−/− and Mlh3−/− males, although specificity of enrichment was confirmed by PCR of random genomic sequences (β-actin) using the same immunoprecipitates (Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200506170/DC1).


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)

Chromatin immunoprecipitation of centromere-associated repeat sequences by antibodies against MMR proteins. (A) Representative slot blot analysis of major and minor satellite repeat sequences (CEN) performed on crude germ cell lysates immunoprecipitated with antibodies MLH3, CREST, or rabbit IgG (Mock control), compared with input DNA. The same slot blot membrane was stripped and reprobed with a B2 SINE probe as a loading control. (B) Quantitation of three centromere ChIP experiments, expressed as a percentage of input DNA + SD. Statistical analysis was performed using unpaired t-tests (***P < 0.0001). (C) ChIP assay followed by PCR using primers against minor and major satellite sequences performed on purified pachytene cells from Pms2−/− testes. Lanes (from left to right): lane 1, molecular weight marker; lanes 2–5, minor satellite PCR; lanes 6–9, major satellite PCR. M, 100 bp marker; Input, input DNA. 10-fold dilutions of ChIP pull-down substrate DNA was used for minor satellite PCR, DNA for major satellite PCR was undiluted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171243&req=5

fig4: Chromatin immunoprecipitation of centromere-associated repeat sequences by antibodies against MMR proteins. (A) Representative slot blot analysis of major and minor satellite repeat sequences (CEN) performed on crude germ cell lysates immunoprecipitated with antibodies MLH3, CREST, or rabbit IgG (Mock control), compared with input DNA. The same slot blot membrane was stripped and reprobed with a B2 SINE probe as a loading control. (B) Quantitation of three centromere ChIP experiments, expressed as a percentage of input DNA + SD. Statistical analysis was performed using unpaired t-tests (***P < 0.0001). (C) ChIP assay followed by PCR using primers against minor and major satellite sequences performed on purified pachytene cells from Pms2−/− testes. Lanes (from left to right): lane 1, molecular weight marker; lanes 2–5, minor satellite PCR; lanes 6–9, major satellite PCR. M, 100 bp marker; Input, input DNA. 10-fold dilutions of ChIP pull-down substrate DNA was used for minor satellite PCR, DNA for major satellite PCR was undiluted.
Mentions: ChIP of mixed germ cell extracts from Pms2−/− males with the anti-MLH3 antibody enriched centromeric major/minor satellite sequences above control levels at comparable rates to that seen for polyclonal CREST serum, which recognizes a variety of CENPs (Fig. 4, A and B). The enrichment of centromere repeats in Pms2−/− spermatocytes with anti-MLH3 antibody or with CREST was statistically elevated compared with mock controls (unpaired t tests, P < 0.0001). No enrichment of similar regions was found in ChIP extracts from Mlh1−/− and Mlh3−/− males, although specificity of enrichment was confirmed by PCR of random genomic sequences (β-actin) using the same immunoprecipitates (Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200506170/DC1).

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