Huan Chen, Michael Lisby, Lorraine S. Symington  Molecular Cell 

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RPA Coordinates DNA End Resection and Prevents Formation of DNA Hairpins  Huan Chen, Michael Lisby, Lorraine S. Symington  Molecular Cell  Volume 50, Issue 4, Pages 589-600 (May 2013) DOI: 10.1016/j.molcel.2013.04.032 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 RPA Is Essential for Both Extensive Resection Pathways (A) Schematic representation of the band disappearance assay for DSB end resection. Red bars show the location of the two probes used for hybridization. (B) Southern blot analysis of the StyI-XhoI-digested genomic DNA from indicated strains taken at different time points after HO induction, showing resection 0.7 and 2.6 kb from the DSB. The bracket indicates the smeared products observed in the td-RFA1 derivatives and the sgs1Δ exo1Δ mutant. (C) Quantification of the 2.4 kb fragment located 2.6 kb from the DSB by the ratio at each time point to 0 hr. The mean values from three independent trials are plotted, and error bars show SD. (D) Schematic representation of the quantitative PCR method to monitor HO-induced DSB end resection. (E) Plot showing the ratio of resected DNA among HO cut DNA at each time point by qPCR analysis. The mean values from three independent trials are plotted, and error bars show SD. See also Figures S1 and S2. Molecular Cell 2013 50, 589-600DOI: (10.1016/j.molcel.2013.04.032) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 MRX-Sae2 and RPA Act Sequentially in End Resection A synergistic defect of td-RFA1 and mre11Δ or sae2Δ in resection was seen by both the band disappearance assay (A) and qPCR (B). Plot showing the ratio of resected DNA (0.7 kb fragment) among the HO cut DNA at each time point by qPCR analysis. The mean values from three independent trials are plotted, and error bars show SD. Molecular Cell 2013 50, 589-600DOI: (10.1016/j.molcel.2013.04.032) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 RPA Is Required to Shield the 3′ ssDNA Tails from Nucleolytic Degradation (A) Both 3′ and 5′ end degradation were observed (R1a) when Rfa1 was depleted. Alkaline electrophoresis of XbaI-digested genomic DNA from different time points after HO induction. ssDNA intermediates were detected by 3′ or 5′ strand-specific RNA probes spanning the region from 0.2 to 0.7 kb distal of the HO cut site. (B) Schematic showing alkaline gel analysis of genomic DNA digested by XbaI. Possible DNA intermediates formed after HO cutting and resection are shown. The black bar shows the location of the probe used. The thin gray lines represent fragments detected by 3′ strand-specific probe. (C) Only 3′ tails were detected in sgs1Δ exo1Δ and td-RFA1 cells. StyI-digested genomic DNA was run on a neutral gel, transferred without denaturing, and hybridized with the indicated strand-specific probes. See also Figure S3. Molecular Cell 2013 50, 589-600DOI: (10.1016/j.molcel.2013.04.032) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 RPA Is Required for Dna2 but Not for Exo1 Localization to DSBs (A) Epifluorescence microscopy showing DSB induced foci formation of YFP-tagged Rfa1 or td-Rfa1 and Dna2-CFP in G2/M-arrested cells. Arrowheads indicate foci. Scale bar is equal to 3 μm. (B) Quantitation of Dna2-CFP foci. The percentage of cells with Dna2 foci was quantitated for the experiment shown in (A), of which more than 90% colocalized with RPA. (C) Exo1 focus formation is independent of Rfa1. Exo1-YFP and Rfa1-CFP or td-Rfa1-CFP were monitored before (t = 0) and 2 or 3 hr after HO expression in G2-M-arrested cells. The left panel shows Rfa1-CFP cells, and the right panel shows td-Rfa1-CFP cells. For the CFP channel, an 8-fold contrast-enhanced version of the images from td-Rfa1-CFP cells is shown to illustrate that some cells contain weak residual Rfa1 foci that colocalize with Exo1 (t = 2), while other cells exhibit an Exo1 focus even in the complete absence of detectable td-Rfa1-CFP. (D) Quantitation of Exo1 and Rfa1 foci. For the experiment in (C), 200–600 cells were examined for Exo1 and Rfa1 foci at each time point. Error bars indicate 95% confidence intervals. Significance was determined by Fisher’s exact test. See also Figure S4. Molecular Cell 2013 50, 589-600DOI: (10.1016/j.molcel.2013.04.032) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 RPA Prevents the Formation of Fold-Back Structures (A) Schematic showing alkaline gel analysis of genomic DNA digested by StyI. Possible DNA intermediates formed after HO cutting and resection are shown. The red bars show the locations of the probes used. The red lines represent fragments detected by the 3′ strand-specific probe close to the HO cut site. (B) Four different dsDNA probes were used to determine the range of fragments r1b and r2b. (C) Alkaline electrophoresis of the StyI-digested genomic DNA from different time points after HO induction. ssDNA intermediates were detected by the 3′ strand-specific RNA probe. r1b and r2b bands can be eliminated by mung bean nuclease treatment of StyI-digested DNA. (D) Schematic showing the formation of hairpins. Alkaline electrophoresis of the StyI-digested genomic DNA from different mutants showing their contribution to hairpin formation. See also Figures S3 and S5. Molecular Cell 2013 50, 589-600DOI: (10.1016/j.molcel.2013.04.032) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Model for RPA-Mediated Coordination of DNA End Processing Mre11 and Sae2 initiate resection to form short 3′ tails. In the absence of RPA, Dna2 is not recruited to the break site, extensive resection is eliminated, and the 3′ overhangs tend to form secondary structures by annealing between short repeats. Following annealing between inverted repeats, the 3′ unpaired flap is trimmed and after DNA synthesis is ligated to the 5′ end to form a hairpin. The 3′ strand loss could occur by Mre11-dependent degradation of 3′ ends or by hairpin cleavage before (shown) or after DNA synthesis and ligation. Molecular Cell 2013 50, 589-600DOI: (10.1016/j.molcel.2013.04.032) Copyright © 2013 Elsevier Inc. Terms and Conditions