Figure S1: Mei11 Is a Mutant Allele of Dmc1
(A) Positional cloning of Mei11. Schematic representation of mouse Chr 15, showing the relative location of MIT markers used in genetic mapping (small circles) and the number of recombinants/total backcross progeny genotyped.
(B) Sequence traces of Dmc1 RT-PCR products. Noncoding strand is shown. Mei11 contains two adjacent nucleotide changes, resulting in double peaks in the heterozygote (marked by bracket; homozygotes cannot be generated due to male sterility).
(C) DMC1 protein structure. A, B, L1, and L2 represent amino acid sequences encoding Walker Motif A, Walker Motif B, and disordered loop regions L1 and L2, respectively. The asterisk indicates the location of the A272 > P mutation in DMC1Mei11, which encodes the amino acid immediately proximal to the L2 loop.
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Figure S2: Mutant Zygotene-Stage Spermatocyte Nuclei Contain Normal Levels of RAD51 Foci
Wt (A) and mutant (B–C; of indicated genotypes) spermatocyte surface–spread nuclei were colabeled with antibodies against SYCP3 (red) and RAD51 (green). +, wt; −, Dmc1−; Mei11, Dmc1Mei11.
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Figure S3: Decreased DMC1 Foci in Mutant Spermatocytes
Surface-spread spermatocyte chromosomes were colabeled with antibodies against STAG3 (a component of the meiotic cohesin core that coincides with lateral elements of the SC) and DMC1. +, wt; −, Dmc1−; Mei11, Dmc1Mei11.
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Figure S4: Dmc1Mei11 Mutant Females Have Reduced Numbers of Primordial and Primary Follicles
The graph shows ovarian follicle counts (y-axis) from both ovaries of 6-wk-old mutant and wt control littermates as described in Materials and Methods. The types of follicles are indicated on the x-axis. Mei11 = B6− Dmc1Mei11/+ (n = 5 ovaries from three females; shown is the average/ovary); Mei11/− = B6− Dmc1Mei11/Dmc1− (n = 2 ovaries from one female); wt = C57BL/6J (n = 6 ovaries from three females). Error bars are standard error of the mean.
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Figure S5: Synaptic Defects, Core Fragmentation, and Persistent DSBs in Mutant Oocytes with >40 Meiotic Cores
(A–B) Mutant oocytes with >40 meiotic cores showing a lack of synapsis.
(C) Wt oocyte nucleus with fully synapsed chromosomes.
(D) Core fragmentation in mutant as suggested by lack of CREST staining (white arrow denotes fragmented core lacking centromeric CREST staining).
(E) Persistent DSBs as identified by persistent RAD51 foci.
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Figure S6: The Mei11 Mutant Allele Does Not Disrupt DMC1 Homotypic Protein Interactions
Shown are yeast two-hybrid interactions of mouse wt and DMC1Mei11 proteins, with clones and yeast strains constructed as described in Materials and Methods. All strains were grown on SD-Leu/Trp/His/Ade agar plates. 1–2: DMC1 bait/DMC1Mei11 prey; 3–4: DMC1 bait/DMC1prey; 5–6: DMC1Mei11 bait/DMC1Mei11 prey.
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Figure S7: DMC1Mei11 Does Not Affect the Ability of DMC1 to Form D-Loops
(A) The ability of human DMC1 (HuDMC1) to form D-loops was analyzed in the presence of varying amounts of HuDMC1Mei11 (A272P). HuDMC1 and A272P were incubated together in the indicated ratios (1 mM total protein concentration) with ssDNA, followed by the addition of dsDNA. For mock treatments, an identical volume of A272P-free buffer was added to the reaction (these contained decreasing amounts of wt HuDMC1, identical to that in the WT:A272P lanes).
(B) Graphs of data from experiments of the type shown in (A). These represent an average of four independent experiments.
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Table S1: Fecundity of Dmc1Mei11/+ Females and Sterility of Dmc1Mei11/Dmc1− Females
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