Transient Stability of DNA Ends Allows Nonhomologous End Joining to Precede Homologous Recombination  Marie Frank-Vaillant, Stéphane Marcand  Molecular Cell  Volume 10, Issue 5, Pages 1189-1199 (November 2002) DOI: 10.1016/S1097-2765(02)00705-0

Figure 1 Quantification of Unprocessed DNA Ends Generated by HO Using a Ligation-Mediated PCR (A) Schematic representation of the LM-PCR assay. (B) Positions of the primers used in this assay. (C) LM-PCR before and after 1 hr of HO induction by galactose in dnl4Δ mutant strain Lev349 growing exponentially in raffinose. Primer and genomic DNA concentrations for the ligation were 50 nM and 5 ng/μl, respectively. Molecular Cell 2002 10, 1189-1199DOI: (10.1016/S1097-2765(02)00705-0)

Figure 2 Specificity of the LM-PCR for Unprocessed DNA Ends (A) HO was induced 1 hr in dnl4Δ mutant strain Lev349 growing exponentially. A fixed amount of genomic DNA (5 ng/μl) was ligated with successive 2-fold dilutions of primers with different 3′ overhangs. Ligation products and a control region at LYS2 were coamplified by PCR. The relative signal between the LM-PCR product and the LYS2 PCR product was calculated for each lane by quantification with an InstantImager. (B) Sequence and position of the BstXI site at MAT. HO was induced 1 hr in dnl4Δ mutant strains Lev349 (MATa) and Lev473 (MATa-BstXI) growing exponentially in raffinose. Genomic DNA was diluted in restriction buffer with (+) or without (−) BstXI and incubated 2 hr at 50°C. PCR with primers on each side of the HO or BstXI site produced a 501 bp fragment and indicated cleavage efficiency (upper gel). LM-PCR was carried out with primer A at 50 nM (lower gel). The relative signal between the two PCR products was calculated for each lane by quantification with an InstantImager. Molecular Cell 2002 10, 1189-1199DOI: (10.1016/S1097-2765(02)00705-0)

Figure 3 Transient Stability of DNA Ends Generated by HO Cells from dnl4Δ mutant strain Lev349 growing exponentially in raffinose were synchronized in G1 with α factor (at 10−6 M) or in G2 with nocodazole (at 15 μg/ml). After 4 hr, HO was induced continuously with 2% galactose. After 30 min, half of the G1-blocked culture was released in S phase by three washes in galactose containing medium. (A) FACS analysis of DNA content. (B) The relative signals between the LM-PCR product and the LYS2 PCR product were normalized to the point of maximum value. Molecular Cell 2002 10, 1189-1199DOI: (10.1016/S1097-2765(02)00705-0)

Figure 4 End Stability in Mutants Defective for NHEJ Cells from strains Lev348 (wt), Lev349 (dnl4Δ), Lev379 (lif1Δ), Lev406 (lif2Δ), Lev402 (yku70Δ), and Lev370 (yku80Δ) growing exponentially in raffinose were either synchronized in G1 with α factor (A) or maintained in exponential growth (B), and induced continuously for HO with 2% galactose. The relative signals between the LM-PCR product and the LYS2 PCR product were normalized to the point of maximum value. Molecular Cell 2002 10, 1189-1199DOI: (10.1016/S1097-2765(02)00705-0)

Figure 5 Crosslinked Chromatin Immunoprecipitation Analysis of Yku70p, Yku80p, and Rad52p In Vivo Interaction with a DNA End Generated by HO Cells from strains Lev348 (untagged), Lev359 (Yku70p-myc), Lev355 (Yku80p-myc), Lev393 (Yku80p-myc yku70Δ), and Lev367 (Rad52p-myc) were grown exponentially in raffinose. Half of the cultures were induced for HO with 2% galactose for 1 hr. (A) Amplifications of the sequence immediately adjacent to the HO break (upper gel) and of the ligation product obtained with primer A (lower gel) are shown. Amplification of a LYS2 sequence is an internal control. (B) The ratio between the MAT signal and the LYS2 control signal were normalized to the induced point obtained with the untagged strain Lev348. Molecular Cell 2002 10, 1189-1199DOI: (10.1016/S1097-2765(02)00705-0)

Figure 6 Competition between NHEJ and HR to Repair an HO Break (A) Schematic representation of the assay for the repair of an HO break at MAT. MATa-inc is an uncleavable MATa sequence with a 2 base pair insertion at the HO cleavage site. (B) Cells from strains Lev348 (wt) and Lev349 (dnl4Δ) bearing a control vector pRS316 or plasmid sp403 (MATa-inc) were grown exponentially in raffinose, synchronized in G1 with α factor, and induced for HO 1 hr with galactose. Cells were washed, resuspended in glucose containing medium with α factor, and spread on plates immediately or after 1, 2, or 3 hr. The fractions of galactose-sensitive and -resistant cells were calculated by averaging two samples. (C) Schematic representation of the MATa-inc sequence integrated at URA3 on chromosome V. The 3′ end of the primers used to distinguish the repair products by PCR are indicated. Molecular Cell 2002 10, 1189-1199DOI: (10.1016/S1097-2765(02)00705-0)