Volume 11, Issue 6, Pages 1661-1671 (June 2003) Equal Sister Chromatid Exchange Is a Major Mechanism of Double-Strand Break Repair in Yeast  Sergio González-Barrera, Felipe Cortés-Ledesma, Ralf E Wellinger, Andrés Aguilera  Molecular Cell  Volume 11, Issue 6, Pages 1661-1671 (June 2003) DOI: 10.1016/S1097-2765(03)00183-7

Figure 1 Physical Analysis of HO-Induced DSB Repair (A) Monocopy plasmid pRS316-TINV including the recombination substrate TINV. Fragments generated by HO cleavage and XhoI-SpeI digestion are indicated with their corresponding sizes in kb. (B) Kinetics of HO induction and DSB repair. Yeast DNA was isolated from cells grown on either SC-2% glucose (Glu) or after different times in SC-2% galactose medium (Gal), doubly digested with XhoI and SpeI, separated by gel electrophoresis, and analyzed by Southern blotting. The 32P-labeled probe for detecting the LEU2 gene (0.6 kb ClaI-EcoRV fragment) is depicted in Figure 2. Quantification of bands resulting from HO cleavage (HO-X and HO-S, closed symbols) and recombination intermediates (REC 2.9 and 4.7, open symbols) is relative to the total plasmid DNA. Note that the low signal intensity of the HO-X band might have interfered with the correct quantification of this band. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)

Figure 2 Scheme of Putative Recombination Events Initiated by a HO-Induced DSB in Plasmid pRS316-TINV Inversions could occur by intrachromatid break-induced replication (BIR; left) or by unequal sister chromatid exchange (SCE; middle). Whether occurring by reciprocal exchange or BIR, unequal SCE would lead to head-to-head dimers (HHDs) and to 2.9 and 4.7 kb fragments when digested with SpeI and XhoI, whereas equal SCE (right) would lead to head-to-tail dimers (HTDs) and to 3.8 kb SpeI-XhoI fragments. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)

Figure 3 PCR Analyses of TINV Recombination Intermediates and Products (A) Scheme of the TINV inverted repeat region in the parental and inverted configuration and the 2.9 kb SpeI band of the recombination intermediate. Primers (arrows a, b, c, and d) and expected PCR products (gray lines) are shown. (B) PCR analysis of genomic DNA isolated from yeast cells grown for 12 hr in 2% galactose. PCR was performed with different primer combinations (see [A]). Band sizes of marker DNA (GIBCO-BRL, 1 kb-plus ladder) are indicated. The incapacity to detect a PCR product with primers a and c at time 0 indicates that all inversions detected were DSB induced. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)

Figure 4 Southern Analysis of Undigested pRS316-TINV and pCM189-leu2HOr and In-Gel Digested pRS316-TINV (A) Diagram of the different type of monomers and dimers expected after induction of HO cleavage in pRS316-TINV. Fragments generated by HO cleavage and XhoI digestion are indicated with their corresponding sizes in kb. (B) Kinetics of HO-mediated cleavage and multimer formation in 2% galactose in plasmid pRS316-TINV. Total DNA was digested with BglII (BglII restriction sites are not present in pRS316-TINV) and separated by electrophoresis in a 0.8% agarose gel. “X” depicts two bands that are barely distinguishable. Since the upper of the two bands was present before HO-induction, only the lower was considered to consist of multimers (tetramers, see text for explanation). Note that linear dimers migrate together with relaxed monomers. D and X band values were normalized with respect to total plasmid DNA. (C) 2D agarose gel analyses of pRS316-TINV dimer formation. DNA isolated from cells grown for 24 hr in SC-2% glucose (no HO-induction) or SC-2% galactose (HO-induction) was separated by size in a 0.8% agarose gel. After EtBr staining, the gel slice of interest was cut out and incubated without (−XhoI) or with (+XhoI) restriction enzyme. After electrophoresis of the cleavage products in the second dimension gel, Southern blot analysis was performed with a 0.6 kb ClaI-EcoRV LEU2 probe. Black arrows mark the DNA fragments resulting from cleavage of dimers (see [A]). The fraction of plasmids that remained uncut as a consequence of partial in-gel digestion is observed as either relaxed or supercoiled molecules. A star identifies an unspecific signal of the membrane. For technical reasons the nondigested (−XhoI) slow migrating plasmid multimers were cut off the gel prior to Southern blot. (D) Kinetics of HO-mediated DSBs and dimer formation in plasmid pCM189-leu2HOr after HO activation in SC-2% galactose. DNA was digested with HaeI (HaeI restriction sites are not present in pCM189-leu2HOr). Other details as in (B). Relaxed dimers (rD), relaxed monomers (rM), supercoiled dimers (scD), linear monomers (lM), supercoiled monomers (scM), and multimers (X) are indicated. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)

Figure 5 Physical Analysis of HO-Induced SCE in rad51Δ and rad59Δ Mutants (A) Scheme of plasmid pRS314-TU3 and structure of the expected plasmid dimer generated by unequal SCE. Bands expected by SpeI-XhoI digestion are shown with their corresponding sizes in kb. (B) Kinetics of HO-induced recombination as determined by Southern analysis. Quantification of the HO-induced DSB (HO-S 3.5 kb, closed symbols) and the 4.0 kb recombination product (REC 4.0 kb, open symbols). Asterisks indicate unspecific hybridization. The 2.2 kb band results from cross-hybridization with the endogenous leu2ΔSFA1. For details see Figure 1. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)

Figure 6 Kinetics of HO Induction and DSB Repair in an mre11Δ Strain Using Plasmid pRS316-TINV Wild-type data are taken from Figure 1. Other details as in Figure 1. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)

Figure 7 Genetic Analysis of Leu+ Inversions (A) Frequency of HO-induced Leu+ recombinants and percentage of inversions in wild-type, rad51Δ, rad59Δ, rad51Δrad59Δ, and rad52Δ cells. HO-induced Leu+ frequency values are the median value obtained from 3–4 independent recombination tests (bottom numbers). The percentage of noncrossovers (dashed) and inversions (white) was determined from 42–84 independent Leu+ recombinants in each strain analyzed. (B) PCR and Southern analyses of 20 independent HO-induced Leu+ recombinants. PCR was performed using a mix of three primers (a, b, and c). The 2.16 and 1.95 kb fragments correspond to the parental and inverted configurations as shown in Figure 3A. For the Southern analysis, total yeast DNA was digested with SspI. The 2.03 and 3.67 kb fragments correspond to the parental configuration, whereas the 1.47 and 4.23 kb bands correspond to the inverted configuration. A 1.2 kb ClaI-SspI LEU2 internal fragment was used as a probe. In all cases the bands obtained coincided with the expected pattern of either inversions or noninversions according to the PCR analysis. In few cases, the presence of both parental and inverted configuration could be detected, indicating the presence of two different plasmids. The 1.6 kb and 2.0 kb marker bands (L) are shown. Molecular Cell 2003 11, 1661-1671DOI: (10.1016/S1097-2765(03)00183-7)