1. Volume 17, Issue 12, Pages 3359-3368 (December 2016)
RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication 
Patrick Ruff, Roberto A. Donnianni, Eleanor Glancy, Julyun Oh, Lorraine S. Symington 
Cell Reports 
Volume 17, Issue 12, Pages (December 2016)
DOI: /j.celrep
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2. Cell Reports 2016 17, 3359-3368DOI: (10.1016/j.celrep.2016.12.003)
Copyright © 2016 The Author(s) Terms and Conditions

3. Figure 1 The rfa1 Mutants Are Proficient in Intra-chromosomal HR
(A) Schematic of the MAT locus showing the location of the HO cut site and the StyI restriction sites used to distinguish between MATa and MATα alleles. The red line indicates the location of the probe used.
(B) Genomic blot of DNA extracted from the indicated strains at different times after HO induction.
(C) Cartoon of the direct repeat recombination reporter. After I-SceI cleavage repair can occur by gene conversion (GC), retaining the TRP1 marker between the repeats, or by SSA resulting in deletion of TRP1 and one of the repeats. Repair products are mostly Ade+.
(D) The frequency of GC and SSA repair of the indicated strains is shown. WT, wild-type. Error bars indicate SD (n = 4–6). ∗p < 0.05.
See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2 BIR Is Defective in rfa1 Hypomorphic Mutants
(A) Schematic of the ectopic BIR assay. The locations of the primers used to monitor DSB formation (D1 and D2), BIR product (P1 and P2), and control primers (C1 and C2) are indicated by horizontal arrows.
(B) BIR frequencies of the indicated strains with the donor cassette located 15, 70, or 128 kb from the telomere. Error bars show SD (n = 3–5).
(C) Detection of BIR intermediates by PCR. Error bars show SD (n = 3).
(D) Schematic showing the predicted sizes of genomic EcoRV fragments before and after BIR.
(E) Genomic blot of the indicated strains showing formation of completed BIR product in real time.
See also Figures S2 and S3.
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5. Figure 3
The rfa1-S373P Mutant Is Defective for Long-Tract Gene Conversion
(A) Schematic showing the recipient LEU2 gene targeted by HO on Chr V (black line) and the donor cassettes on Chr III (gray lines) with varying distance between LE and U2.
(B) Percent survival of the indicated strains. Error bars show SD (n = 3).
Cell Reports  , DOI: ( /j.celrep )
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6. Figure 4
Rad51 Overexpression, but Not mph1Δ, Suppresses the rfa1 BIR Defect
(A) Percent BIR for the indicated strains expressing empty vector (EV) or RAD51 from a high-copy-number vector. Error bars show SD (n = 3).
(B) Rad51 enrichment at sequence 2 kb upstream of the DSB in the indicated strains 0, 2, or 4 hr after HO induction. Error bars show SD (n = 3).
(C) Percent repair for the indicated 5 kb gap strains expressing EV or RAD51 from a high copy number vector. Error bars show SD (n = 3).
(D) Percent BIR for the indicated MPH1 and mph1Δ strains. Error bars show SD (n = 3).
See also Figures S4 and S5.
Cell Reports  , DOI: ( /j.celrep )
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7. Figure 5
3′ Strand Loss Contributes to the BIR Defects of the rfa1 Mutants
(A) Fraction of recipient locus retained in the indicated strains at 2, 6, and 10 hr after HO induction.
(B) Percent BIR for the indicated strains. Error bars show SD (n = 3). ∗p < 0.05; ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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8. Figure 6 Model for Break Repair and BIR
Break repair involves short ssDNA intermediates that are less susceptible to degradation when RPA is dysfunctional. By contrast, BIR involves long-lived ssDNA intermediates formed by end resection and migrating D-loop synthesis that are more dependent on fully functional RPA for protection from degradation. Naked ssDNA is prone to base damage or hydrolysis and forms secondary structure that could be targeted by structure-selective nucleases, thereby destroying recombination intermediates. Rad51 OE suppresses the BIR defect of the rfa1 mutants, pre or post-synaptically, by protecting ssDNA and promoting strand invasion.
Cell Reports  , DOI: ( /j.celrep )
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