1. Intermediates of Yeast Meiotic Recombination Contain Heteroduplex DNA
Thorsten Allers, Michael Lichten 
Molecular Cell 
Volume 8, Issue 1, Pages (July 2001)
DOI: /S (01)

2. Figure 1 Ectopic Recombination System
(A) Map of his4::URA3-arg4-EcPal9 (his4::arg4-pal) and leu2::URA3-ARG4 (leu2::ARG4) inserts and the probes used. The EcoRI site-containing palindrome (at +9 of the ARG4 coding region) is indicated by a lollipop. Meiotic DSBs occur at two sites (DSB1 and 2, vertical arrows) upstream of the URA3 (gray box) and ARG4 (black box) open reading frames. Crossovers between inserts may occur upstream (left) or downstream (right) of the palindromic marker. HIS4 and LEU2 are 16.7 kb (14.0 cM) apart on chromosome III. E, EcoRI; P, PvuII.
(B) Segregation of arg4-EcPal9 and associated crossovers in MJL2442. Eight-spored fungal nomenclature is used (i.e., a 3+:1– tetrad is designated 6+:2–). Incidental: the converted allele is on a noncrossover chromatid in a tetrad that contains a crossover; indeterminate: crossover intervals cannot be mapped in tetrads with a full arg4-EcPal9 conversion; other non-PMS: two 8+:0– tetratypes; other PMS: one 7+:1– nonparental ditype and one noncrossover aberrant 4+:4–, each counted as two independent events. Nonparental ditype tetrads (4) are also counted as two single events
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 Joint Molecules Formed During Meiosis
(A) EcoRI digestion reveals three JM species. An EcoRI (E) digest of meiotic DNA from MJL2590 (10 hr sample) was displayed on a native/native 2D gel and probed with sequences upstream of the arg4-EcPal9 palindrome (ARGU). URA3-ARG4 sequences are shown in black, and LEU2 and HIS4 sequences are shown in red and blue, respectively. Holliday junctions in JM1 are shown as predicted by the DSBR model, but could be located elsewhere. A faint arc of Y-shaped replication or recombination intermediates (R) is also observed as is an unidentified species (asterisk). The 1.8 kb his4::arg4-pal fragment migrated off the gel and is not seen here.
(B) Assay for hDNA. Digestion with EcoRI (E) and PvuII (P) yields a 6.9 kb leu2::ARG4 parental fragment and 1.8 kb and 3.0 kb his4::arg4-pal parental fragments. When in hDNA, the palindrome assumes a hairpin configuration; digestion with EcoRI nicks the strand carrying the palindrome. Recombinant his4::arg4-pal molecules that have lost the palindrome from one or both strands generate a novel 4.8 kb molecule.
(C) Detection of hDNA on native/denaturing 2D gels. The first (native) dimension resolves molecules based on duplex size; JMs migrate above the linear species. In the second dimension, duplexes and JMs are denatured into their component strands: 6.9 kb and 1.8 kb for hDNA at leu2::ARG4, 4.8 kb and 1.8 kb for hDNA at his4::arg4-pal. The 1.8 kb strands (underlined) that migrate off the diagonal of parental DNA are therefore diagnostic of hDNA.
(D) hDNA and JM strands. An EcoRI/PvuII digest of the same DNA sample was displayed on a native/denaturing 2D gel and probed with ARGU. On-diagonal 6.9 kb (leu) and 1.8 kb (his) spots contain parental sequences. Off-diagonal spots (1.8 kb; h) are diagnostic of hDNA; the 4.8 kb on-diagonal spot (g) is diagnostic of palindrome loss from his4::arg4-pal. Component strands of JMs migrate within the area bounded by dashed lines. JM1 (11.7 kb) contains parental leu2::ARG4 and his4::arg4-pal strands and nonparental 4.8 kb his4::arg4-pal strands. JM2 (9.9 kb) contains parental leu2::ARG4 strands but is missing his4::arg4-pal strands due to loss of the upstream EcoRI fragment. JM3 (8.7 kb) contains parental strands of both leu2::ARG4 and his4::arg4-pal inserts
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3 Strand Analysis of Joint Molecules
An EcoRI/PvuII digest of meiotic DNA from MJL2590 (10 hr sample) was displayed on native/denaturing 2D gels. Only the relevant portions are shown (dashed lines in Figure 2D; entire images supplied upon request). Filters were probed with strand-specific probes to flanking LEU2 and HIS4 sequences, both upstream (leuU and hisU) and downstream (leuD and hisD) of the insert.
(A) JM1 contains hDNA. Both leu2::ARG4 strands (red arrows) are parental in length (6.9 kb). Amongst his4::arg4-pal strands (blue arrows), the 5′-3′ strand has lost the palindrome (4.8 kb), and the 3′-5′ strand has retained it (1.8 and 3.0 kb). The inferred structure of JM1 is shown; Holliday junctions could be located elsewhere in the region of homology. Asterisks denote JMs produced by sister-chromatid recombination between insertless LEU2 genes.
(B) JM2 contains parental leu2::ARG4 (6.9 kb) and his4::arg4-pal (3.0 kb) strands joined by Holliday junctions downstream of the palindrome. The upstream EcoRI fragment is absent (1.8 kb; indicated by brackets), so JM2 is not detected by hisU probes.
(C) JM3 contains parental leu2::ARG4 (6.9 kb) and his4::arg4-pal (1.8 kb) strands joined by Holliday junctions upstream of the palindrome. The downstream EcoRI fragment is absent (3.0 kb; indicated by brackets), so JM3 is not detected by hisD probes
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 Proposed Mechanisms
(A) The double-strand break repair model. A DSB is resected to expose 3′-ended single-strand tails, which invade the homolog. A potential mismatch is indicated by a lollipop. DNA synthesis and ligation generate a double Holliday junction intermediate with hDNA flanking the DSB site. Cutting the junctions as shown yields a downstream crossover associated with hDNA at the site of the marker.
(B) Bubble migration. Resection of the DSB does not extend to the palindrome. A double Holliday junction forms upstream of the marker, which is in parental configuration. Migration of both Holliday junctions past the palindrome yields a JM2 intermediate and, thus, a downstream crossover with little upstream hDNA.
(C) Strand displacement-mediated crossing-over. Strand invasion and synthesis occur on only one side of the DSB site. The invading strand is partially displaced and reanneals with the broken chromosome. The remaining D-loop is transformed into a double Holliday junction. DNA synthesis restores the marker to parental configuration, and resolution of the double Holliday junction yields a downstream crossover without associated hDNA at the marker
Molecular Cell 2001 8, DOI: ( /S (01) )