Comprehensive, Fine-Scale Dissection of Homologous Recombination Outcomes at a Hot Spot in Mouse Meiosis  Francesca Cole, Scott Keeney, Maria Jasin  Molecular Cell  Volume 39, Issue 5, Pages 700-710 (September 2010) DOI: 10.1016/j.molcel.2010.08.017 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 39, 700-710DOI: (10.1016/j.molcel.2010.08.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Recombination Initiation Bias for COs and NCOs in A×D Results in Transmission Distortion (A) PCR strategy to amplify COs and NCOs at the A3 hot spot. Only one orientation is shown. Filled circles, polymorphisms on the A (A/J, blue) or D (DBA/2J, red) chromosome; filled arrows, allele-specific primers; open arrows, universal primers (U). (B) CO breakpoints cluster in a 1.5 kb region, defining the A3 hotspot. (i) Total CO breakpoint map is shown. (ii) The CO breakpoint maps in the A-to-D (top) and D-to-A (bottom) orientations are shifted relative to each other, indicating reciprocal CO asymmetry. Vertical lines represent the midpoint for each orientation, as determined from the cumulative distributions. (iii) The distance between the midpoints provides an estimate of the mean CO gene conversion tract length (500 bp, arrow). The vertical line in (iii) represents the average midpoint of CO gene conversion tracts, as determined from the midpoints for the two orientations. Numbers of COs examined and Poisson-adjusted CO frequencies (± SD) are indicated. Ticks represent positions of the 20 tested polymorphisms. Red circles, indels. See Figures S2D and S2E. (C) Transmission distortion from COs in favor of the A chromosome arising from preferential DSB formation on the D chromosome (red), with the A chromosome (blue) serving as the donor of genetic information for DSBR. Only one chromatid from each homolog is shown for simplicity. The vertical lines represent the midpoints of the CO distributions for each orientation, as in (Bii). (D) NCO gene conversion tracts on the D (top) and A (bottom) chromosomes. (E) NCO frequencies for A×D are 10-fold higher than CO frequencies, with the D chromosome showing 9-fold more NCOs than the A chromosome. Total Poisson-adjusted NCO frequencies are indicated. NCO frequencies at each tested polymorphism are normalized for coconversions. Ticks in the center represent the 19 polymorphisms tested. The vertical line represents the average midpoint of CO gene conversion tracts, as in (Biii). (F) Transmission distortion in favor of the A chromosome derived from NCOs (gray bars) and COs (blue bars, derived from Figure 1C). Transmission distortion from NCOs arises from preferential DSB formation on the D chromosome (red), with the A chromosome (blue) serving as the donor of genetic information for SDSA. Molecular Cell 2010 39, 700-710DOI: (10.1016/j.molcel.2010.08.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 B×D Shows CO Refraction, but Not Biased Recombination Initiation (A) CO breakpoint maps from B×D in the B-to-D (top) and D-to-B (bottom) orientations. The CO refractory zone is shaded. Ticks represent positions of the 16 tested polymorphisms. (B) Cumulative CO distributions for all COs and for each orientation. (C) NCO frequencies for the D (top) and B (bottom) chromosomes indicate similar frequencies of recombination initiation on both chromosomes. Ticks in the center represent the polymorphisms tested (20 and 16 for the D and B chromosomes, respectively). The vertical line represents the midpoint of the total CO distribution, as in (B). (D) NCO gene conversion tracts on the D chromosome, depicted as in Figure 1D. See Figure S4 for the B chromosome. Molecular Cell 2010 39, 700-710DOI: (10.1016/j.molcel.2010.08.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 CO Refraction Is Linked to Chr 1 and Is Associated with a Wider NCO Distribution (A–D) Total CO breakpoint maps for the indicated hybrids. The number of CO breakpoints mapped to the regions indicated by brackets is given. The leftmost of these regions encompasses the CO refractory zone in B×D, except in (D), where it encompasses the slightly shifted CO refractory zone in C×D. Breakpoints within the CO refractory zone are significantly reduced for B×D and C×D relative to either A×D or A1/B×D; p < 0.0001 (Fisher's exact test). Arrows, midpoints of total CO breakpoints for each hybrid; red circles, indels; black circle, SNP2425. (E) Schematic of polymorphisms for the A, B, or C versus D haplotypes in the area of the CO refractory zone. The three indels and the SNPs mentioned in the text are labeled, with the location of the ∼140 bp inverted repeat indicated (arrows). Polymorphisms of the D genotype are shown in gray; SNPs differing from D are in black, and insertions are in red. The CO refractory zones for B×D and C×D are shown with shading on the B and C chromosomes, respectively. (F) Comparison of NCO distributions for the D chromosome in the indicated hybrids. Only polymorphisms (ticks) shared by all three hybrids are plotted. NCOs are expressed as a percent of total and normalized for coconversions. (G) Lorentzian fit of the NCO distributions (F) showing an increased width for B×D and C×D compared with A×D. A slight shift to the left is observed for the C×D distribution. The amplitude, width, and the center of the fitted distributions are indicated. Molecular Cell 2010 39, 700-710DOI: (10.1016/j.molcel.2010.08.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Coconversions in NCOs Are Enriched in the Central 100 bp of the Hot Spot (A and B) Compilation of NCOs with conversion of only a single polymorphism (singletons) (A) and coconversions (B) for A3, plotted as a percent of total NCOs. Values are Poisson adjusted. The 100 bp zone enriched for coconversions is indicated by the shading. NCOs are derived from all chromosomes tested except the A chromosome. (B) CO breakpoint cumulative distributions (right y axis) for the indicated hybrids are also plotted; the arrow indicates the midpoint for all of the CO breakpoints. (C) Comparison of the frequency of coconversion within and outside of the coconversion zone. Intervals of similar sizes are compared for the number of coconversions relative to total conversions involving one or both polymorphisms. Interval sizes within the coconversion zone may differ slightly between hybrids because of polymorphism variation. Coconversions outside and within the coconversion zone were compared using a Fisher's exact test. Molecular Cell 2010 39, 700-710DOI: (10.1016/j.molcel.2010.08.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 CO Refraction Models (A) Spo11-generated DSB formation is inferred to occur most often to the right of the CO refractory zone (shading). Strand invasion generates heteroduplex DNA encompassing the imperfect inverted repeat (arrows), with indel-2 near the center. DNA repair synthesis causes torsional stress, resulting in cruciform extrusion. (B) Heteroduplex rejection favors NCOs at the expense of COs. Recognition of the aberrant secondary structure by a helicase promotes dissociation of the invading strand and leads to SDSA, generating an NCO. (C) Nuclease cleavage leading to double-strand gap formation. Cleavage by a structure-specific nuclease, followed by dissociation, results in a double-strand gap that extends from the base of the inverted repeat to the Spo11 DSB site. Gap repair without reciprocal exchange generates an NCO with a longer gene conversion tract than normal, incorporating multiple polymorphisms (i.e., coconversions). Molecular Cell 2010 39, 700-710DOI: (10.1016/j.molcel.2010.08.017) Copyright © 2010 Elsevier Inc. Terms and Conditions